WO2011075605A2

WO2011075605A2 - Hyperglycosylated interferon variants and methods of use

Info

Publication number: WO2011075605A2
Application number: PCT/US2010/060884
Authority: WO
Inventors: Jin Hong; Lawrence Blatt
Original assignee: Alios Biopharma, Inc.
Priority date: 2009-12-18
Filing date: 2010-12-16
Publication date: 2011-06-23
Also published as: WO2011075605A3

Abstract

Disclosed herein are hyperglycosylated interferon variants, pharmaceutical compositions comprising the hyperglycosylated interferon variants described herein, and method of ameliorating and/or treating diseases and/or conditions with the hyperglycosylated interferon variants described herein.

Description

HYPERGLYCOSYLATED INTERFERON VARIANTS AND METHODS OF USE

RELATED APPLICATIONS

[0001] The present application claims the benefit of U.S. Provisional Application No. 61/287,884, entitled "HYPERGLYCOSYLATED INTERFERON VARIANTS AND METHODS OF USE" filed December 18, 2009 and 61/288,047, entitled "HYPERGLYCOSYLATED INTERFERON VARIANTS AND METHODS OF USE" filed December 18, 2009, both of which are herein expressly incorporated by reference in their entireties, including any drawings.

REFERENCE TO SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled SEQLISTING.TXT, created December 16, 2010, which is 192 Kb in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

BACKGROUND

Field

[0003] The present application relates to the fields of chemistry, biochemistry and medicine. More particularly, disclosed herein are hyperglycosylated interferon variants, pharmaceutical compositions that include one or more hyperglycosylated interferon variants, and methods of treating diseases and/or conditions with one or more hyperglycosylated interferon variants.

Description

[0004] Interferons (IFNs) are natural cell- signaling glycoproteins produced by the cells of the immune system of most vertebrates in response to challenges such as viruses, parasites and cancer cells. Interferons assist the immune response by inhibiting viral replication within host cells, activating natural killer cells and macrophages, increasing antigen presentation to T lymphocytes, and/or increasing the resistance of host cells to viral infection. Interferons are divided into three classes: type I, type II and type ΠΙ. All classes have been implicated as playing a role in fighting viral infections. SUMMARY

[0005] Some embodiments disclosed herein relate to a hyperglycosylated interferon variant of a parent interferon, wherein the parent interferon can be selected from a Type I interferon, a Type II interferon, and a Type ΙΠ interferon, wherein the hyperglycosylated interferon variant can be the parent interferon that has been modified to include a peptide extension inserted at a terminal region, where the peptide extension can be a peptide of 1-200 consecutive amino acids and can include at least two glycosylation sites, where the terminal region can be selected from the group consisting of an amino- terminal region that consists of the first 15 amino acids at the amino-terminus of the parent interferon that excludes any signal peptide in the parent interferon and a carboxy- terminal region that consists of the last 15 amino acids at the carboxy-terminus of the parent interferon. In some embodiments, the peptide extension can include at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten glycosylation sites. In some embodiments, the parent interferon has been further modified to include at least one or more additional glycosylation sites through at least one amino acid substitution or at least one combination of amino acid substitutions in the amino acid sequence of the parent interferon.

[0006] An embodiment disclosed herein relates to a pharmaceutical composition that can include one or more hyperglycosylated interferon variants of a parent interferon disclosed herein, and a pharmaceutically acceptable carrier, excipient, or combination thereof.

[0007] Some embodiments disclosed herein relate to a method of ameliorating and/or treating a fibrotic disorder. Other embodiments disclosed herein relate to a method of ameliorating and/or treating cancer. Still other embodiments disclosed herein relate to a method of inhibiting the growth of a tumor. Yet still other embodiments disclosed herein relate to a method of ameliorating and/or treating a viral infection.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure la-f depicts an amino acid sequence alignment of the human type I interferons (IFN) and the fusion protein of consensus IFN-aConl with human IFN al4 signal peptide (Infergen w A14 Sig). The interferon sequences that are aligned in Figure la-f are: IFN-al (SEQ ID NO: 79), IFN-a2a (SEQ ID NO: 80), IFN-a2b (SEQ ID NO: 81), IFN-a4a (SEQ ID NO: 82), IFN-a4b (SEQ ID NO: 83), IFN-a5 (SEQ ID NO: 84), IFN-a6 (SEQ ID NO: 85), IFN-a7 (SEQ ID NO: 86), IFN-a8 (SEQ ID NO: 87), IFN- alO (SEQ ID NO: 88), IFN-al3 (SEQ ID NO: 89), IFN-al4 (SEQ ID NO: 90), IFN-al6 (SEQ ID NO: 91), IFN-al7 (SEQ ID NO: 92), IFN-a21 (SEQ ID NO: 93), IFN-aH (SEQ ID NO: 94), IFN-al (SEQ ID NO: 95), IFN-aJl (SEQ ID NO: 96), IFN beta 1 (SEQ ID NO: 97), IFN kappa (SEQ ID NO: 98), IFN omega 1 (SEQ ID NO: 99), IFN tau (SEQ ID NO: 100), and Infergen (SEQ ID NO: 101). The majority sequence (SEQ ID NO: 102) is shown above.

[0009] Figure 2 shows the amino acid sequence of human interferon γ (SEQ ID NO: 37).

[0010] Figure 3 shows the amino acid sequences of human interferon lambda: Figure 3A: human interferon lambdal (SEQ ID NO: 40), Figure 3B: human interferon lambda2 (SEQ ID NO: 42), Figure 3C: human interferon lambda3 (SEQ ID NO: 44).

[0011] Figures 4A-D show Coomassie Blue stained protein gel and western blots of various hyperglycosylated variants of the parent interferon alfacon-1 (CIFN), the parent human interferon beta (IFNB), the parent human interferon gamma (IFNG), the parent human interferon alpha 1 (IFN ocl), and the parent human interferons lambdal (IFN λΐ), lambda2 (IFN λ2) and lambda3 (IFN λ3). The interferons shown in Figure 4A are: CIFN-31-102- 138 (SEQ ID NO: 103), CIFN-Nl-31-102-138 (SEQ ID NO: 64), CIFN-N2-31-102-138 (SEQ ID NO: 65), CIFN-N3-31-102-138 (SEQ ID NO: 66), CIFN- N4-31-102-138 (SEQ ID NO: 67). The interferons shown in Figure 4B are: CIFN-102- 138 (SEQ ID NO: 104) and CIFN-102-138-C2 (SEQ ID NO: 63). The interferons shown in Figure 4C are: CIFN-N14(3)-102, CIFN-N14(3)-102-138, CIFN-N14(3)-108-138, and CIFN-N14(3)-108. The interferons shown in Figure 4D are: CIFN-N11-31-102-138, CIFN-N8-31-102-138, CIFN-N6-31-102-138, and CIFN-N4-31-102- 138. Figure 4E shows western blots of CIFN-N11-31-102-138, CIFN-N11-8-102-138 and CIFN-N6-31- 102-138 with (+) or without (-) glycosidase PNGase F treatment. The interferons shown in Figure 4F are: human IFNB-N10 (SEQ ID NO: 54), IFNB-N8 (SEQ ID NO: 53), IFNB-N6 (SEQ ID NO: 52), IFNB-N4 (SEQ ID NO: 51), and IFNB (SEQ ID NO: 97). Figure 4G shows IFNB-N10, IFNB-N8 and IFNB-N6 with (+) or without (-) glycosidase PNGase F treatment. The interferons shown in Figure 4H are: human IFNG (SEQ ID NO: 37), IFNG-N5 (SEQ ID NO: 39) and IFNG-N10 (SEQ ID NO: 38). The interferon shown in Figure 41 is IFN Ocl-NlO. The interferons shown in Figure 4J are: IFN λ1-Ν16, IFN λ2-N16 and IFN λ3-N16.

[0012] Figure 5 shows the HCV replicon activities and interferon specific activities of CIFN and variouis hyperglycosylated variants of the parent CIFN. Figure 5A shows the HCV replicon activities of CIFN and CIFN-N14(3)-102. Figure 5B shows the HCV replicon activities of CIFN-N14(3)-108 and CIFN-N14(3)-102-138. Figure 5C shows the HCV replicon activity of CIFN-N14(3)-108-138. Figure 5D shows the interferon specific activities of CIFN, CIFN-N14(3)-102, CIFN-N14(3)-102-138, CIFN- N14(3)-108, and CIFN-N14(3)-108-138.

[0013] Figure 6 shows the western blot of various hyperglycosylated variants of the parent human interferon beta (IFNB) having an N-terminal peptide extension with 10 glycosylation sites. Variant EPO-N10 has the signal peptide from human EPO (SEQ ID NO: 124), variant CD33-N10 has the signal peptide from CD33 (SEQ ID NO: 123), variant IFNB-N10 has the signal peptide from IFNB (SEQ ID NO: 127), and variant DS- N10 has an artificial signal peptide (SEQ ID NO: 122).

[0014] Figure 7A shows a graph illustrating the IFN activity of mN14-CIFN- 108 in mouse plasma over time after a single subcutaneous injection in mice. Figure 7B shows graphs illustrating the beta2-microglobulin protein level and the induction of OAS1 mRNA in liver in mice over time after a single subcutaneous injection.

[0015] Figure 8 shows Coomassie Blue stained protein gel and western blots of interferon alfacon-1 (CIFN, SEQ ID NO: 521) and several hyperglycosylated variants of CIFN. The interferons shown in Figure 8A are: CIFN and the hyperglycosylated variant CIFN D31N+P33S. The interferons shown in Figure 8B are: CIFN and the hyperglycosylated variant CIFN D31N+L32S+P33T (SEQ ID NO: 537). The interferons shown in Figure 8C are: CIFN and the hyperglycosylated variant CIFN D108N. The interferons shown in Figure 8D are: CIFN and the hyperglycosylated variant CIFN D108N+S110T (SEQ ID NO: 545).

[0016] Figure 9 shows western blots of various fractions of the hyperglycosylated variant CIFN P33N+K101N+D108N+S110T+N144K+V145N (referred herein as "CIFN variant-6," and the parent CIFN. 4-Gly, 3-Gly and 2-Gly denote, respectively, the fractions of the CIFN variant-6 in which 4, 3 and 2 glycosylation sites are glycosylated. Figure 9A shows purified 4-Gly CIFN variant-6, 3-Gly CIFN variant-6, and 2-Gly CIFN variant-6 along with protein molecular weight markers in a protein gel stained by Coomassie Blue. Figure 9B shows western blots of 2-Gly CIFN variant-6 with (+) and without (-) PNGase F treatment. Figure 9C shows 3-Gly CIFN variant-6 and 4-Gly CIFN variant-6 with (+) and without (-) PNGase F treatment.

[0017] Figure 10 shows a graph illustrating the IFN activity of various fractions of the CIFN variant-6 and the parent CIFN in rat plasma over time after a single subcutaneous injection in rats. 4-Gly, 3-Gly and 2 Gly denote, respectively, the fractions of the CIFN variant-6 in which 4, 3 and 2 glycosylation sites are glycosylated. The y-axis is shown in a logarithmic scale.

[0018] Figure 11 shows the primary end point, survivial rate, in a yellow fever virus (YFV)/hamster study of various fractions of the CIFN variant-6 along with positive controls (CIFN and Peg-IFN-CC-2a) and negative control (PBS). 4-Gly and 3-Gly denote, respectively, the fractions of the CIFN variant-6 in which 4 and 3 glycosylation sites are glycosylated. The 4-Gly CIFN variant-6 and Peg-IFN-0C-2a were dosed at once per week for two weeks while 3-Gly was dosed at twice per week for two weeks. Both CIFN and PBS were dosed at once per day for two weeks.

[0019] Figure 12 shows the secondary end points, the ATL levels in blood and the viral load in the liver and the serum, in a yellow fever virus (YFV)/hamster study of various fractions of the CIFN variant-6 along with positive controls (CIFN and Peg- IFN-0C-2a) and negative control (PBS). 4-Gly and 3-Gly denote, respectively, the fractions of the CIFN variant-6 in which 4 and 3 glycosylation sites are glycosylated. Figure 12A shows changes in the ATL level in blood. Figure 12B shows the viral load in the liver. Figure 12C shows the viral load in the serum. ***, ** and * reprensents P<0.001, P<0.01 and P<0.05, respectively, as compared with the phosphate-buffered saline (PBS) control. The dashed lines in Figures 12B-C represent the limit of detection.

DETAILED DESCRIPTION

Definitions

[0020] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. All patents, applications, published applications and other publications referenced herein are incorporated by reference in their entirety unless stated otherwise. In the event that there are a plurality of definitions for a term herein, those in this section prevail unless stated otherwise. [0021] As used herein, the term "hyperglycosylated interferon variant of a parent interferon" refers to an interferon variant that includes one or more additional glycosylation sites that are not present in a parent interferon. In some embodiments, the parent interferon has been modified to include a peptide extension inserted at a terminal region, where the peptide extension includes one or more glycosylation sites. In some embodiments, the parent interferon has been modified to include (1) one or more additional glycosylation sites in the amino acid sequence of the parent interferon, wherein the additional glycosylation site(s) are introduced by an amino acid substitution and/or a combination of amino acid substitutions, and (2) a peptide extension inserted at a terminal region, where the peptide extension includes one or more glycosylation sites. In some embodiments, the parent interfern has been modified to include one or more additional glycosylation sites in the amino acid sequence of the parent interferon, wherein there is no peptide extension inserted at any of the terminal region of the parent interferon. Each added glycosylation site may be an N-linked glycosylation site or an O-linked glycosylation site.

[0022] As used herein, the term "native glycosylation site" refers to a glycosylation site that exists in a first naturally-occurring interferon and is introduced into a second naturally-occurring or a non-naturally occurring interferon at a homologous amino acid position. A native glycosylation site can exist in one or more naturally- occurring interferons, and the native glycosylation site can be glycosylated or non- glycosylated in the first naturally-occurring interferon. For example, the glycosylation site N-L-S at amino acid positions 31, 32 and 33 of Interferon ocl4 can be introduced at amino acid positions 31, 32 and 33 of interferon alfacon-1 (D31-L32-P33) as a native glycosylation site.

[0023] As used herein, the term "non-native glycosylation site" refers to a glycosylation site that does not exist in any naturally-occurring interferon, as well as a glycosylation site that exists in a first naturally-occurring interferon and is introduced into a second naturally-occurring or a non-naturally occurring interferon at a non-homologous amino acid position. For example, the glycosylation site (N31-L32-S33) at amino acid positions 31, 32 and 33 of interferon ocl4 can be introduced at amino acid positions 108, 109 and 110 of interferon alfacon-1 (D108-E109-S110) as a non-native glycosylation site. As another example, the glycosylation site (N102-S103-S104) at amino acid positions 102, 103 and 104 of interferon ocl4 can be introduced to a peptide extension that is inserted at the N-terminal region of interferon alfacon-1 as a non-native glycosylation site.

[0024] As used herein, non-native and native glycosylation sites include relinked glycosylation sites and O-linked glycosylation sites. N-linked glycosylation sites include, for example, Asn-X-Ser/Thr (N-X-S/T), where the Asparagine (Asn) residue provides a site for N-linked glycosylation and X is any amino acid residue. O-linked glycosylation sites include at least one serine or threonine residue. A number of O-linked glycosylation sites are known in the art and have been described in, for example, Ten Hagen et al., J. Biol. Chem., 1999, 274(39):27867-27874; Hanisch et al., Glycobiology, 2001, 11:731-740; and Ten Hagen et al., Glycobiology, 2003, 13:1R-16R.

[0025] Standard techniques can be used to determine whether an interferon comprises N-linked and/or O-linked glycosylation. See, for example, R. Townsend and A. Hotchkiss, eds., Techniques in Glycobiology, Marcel Dekker Inc., 1997; and E. Hounsell, ed. Glycoanalysis Protocols (Methods in Molecular Biology, Vol. 76), Humana Press, 1998. For example, the change in electrophoretic mobility of a protein before and after treatment with chemical or enzymatic deglycosylation can be used to determine the glycosylation status of a protein. Enzymatic deglycosylation can be carried out using any of a variety of enzymes, including, but not limited to, peptide-N4-(N-acetyl-P-D- glucosaminyl) asparagine amidase (PNGase F), endoglycosidase Fl, endoglycosidase F2, endoglycosidase F3, a(2→3,6,8,9) neuraminidase, and the like. For example, sodium docecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the protein, either pre-treated with PNGase F or untreated with PNGaseF, can be conducted. A marked decrease in band width and change in migration position after treatment with PNGaseF is considered diagnostic of N-linked glycosylation. The carbohydrate content of a glycosylated protein can also be detected using lectin analysis of protein blots (for example, proteins separated by SDS-PAGE and transferred to a support, such as a nylon membrane). Lectins, carbohydrate-binding proteins from various plant tissues, have both high affinity and narrow specificity for a wide range of defined sugar epitopes found on glycoprotein glycans. See Cummings, Methods in Enzymol, 1994, 230:66-86. Lectins can be detectably labeled (either directly or indirectly), allowing detection of binding of lectins to carbohydrates on glycosylated proteins. For example, when conjugated with biotin or digoxigenin, a lectin bound to a glycosylated protein can be easily identified on membrane blots through a reaction utilizing avidin or anti-digoxigenin antibodies conjugated with an enzyme such as alkaline phosphatase, β-galactosidase, luciferase, or horse radish peroxidase, to yield a detectable product. Screening with a panel of lectins with well-defined specificity can provide considerable information about a glycoprotein's carbohydrate complement.

[0026] The phrase "increased glycosylation" is used herein to indicate increased levels of attached carbohydrate molecules, normally obtained as a result of increased number or better utilization of glycosylation site(s). The increased glycosylation may be determined by any suitable method known in the art for analyzing attached carbohydrate structures. For example, Western blot can be used to determine the amount of attached carbohydrates in a glycosylated interferon.

[0027] An amino acid residue "located close to" a glycosylation site is usually located in position -4, -3, -2, -1, +1, +2, +3, or +4 relative to the amino acid residue of the glycosylation site to which the carbohydrate moiety is attached, in particular in position - 2, -1, +1, +2, such as position -1 or +1, in particular position -1. These positions may be modified to increase glycosylation at the glycosylation site. For example, the modification can be a substitution.

[0028] As used herein, the term "polypeptide" refers to a polymer of amino acids. A polypeptide can be of various lengths. Thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. A polypeptide can be with or without N-terminal methionine residues. A polypeptide may include post-translational modifications, for example, glycosylations, acetylations, phosphorylations and the like. Examples of "polypeptide" include, but are not limited to, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, non-coded amino acids, etc.), polypeptides with substituted linkages, fusion proteins, as well as polypeptides with other modifications known in the art, both naturally occurring and non- naturally occurring.

[0029] As used herein, the terms "polynucleotide" and "nucleic acid molecule" are used interchangeably herein to refer to polymeric forms of nucleotides of any length. Thus, oligonucleotides are included within the definition of polynucleotide. The polynucleotides may contain deoxyribonucleotides, ribonucleotides, and their analogs. Nucleotides may have any three-dimensional structure, and may perform various functions. The term "polynucleotide" includes single-, double-stranded and triple helical molecules. For example, a polynucleotide can be a double- stranded DNA found, inter alia, in linear DNA molecules (for example, restriction fragments), viruses, plasmids, and chromosomes. "Oligonucleotide" generally refers to polynucleotides of between about 5 and about 100 nucleotides of single- or double-stranded DNA. Oligonucleotides are also known as "oligomers" or "oligos," and can be isolated from genes, or chemically synthesized by methods known in the art.

[0030] Non-limiting embodiments of polynucleotides include: genes or gene fragments, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide can also include modified nucleic acid molecules, such as methylated nucleic acid molecules, nucleic acid molecules with modified purine or pyrimidine bases and nucleic acid molecule analogs. Nucleic acids may be naturally occurring DNA or RNA, or may be synthetic analogs of DNA or RNA, such as those known in the art. Synthetic analogs include native structures that have been modified to include alterations in the backbone, sugars and/or heterocyclic bases. Examples of modified backbone include phosphorothioates; phosphorodithioates, where both of the non-bridging oxygens are substituted with sulfur; phosphoroamidites; alkyl phosphotriesters; boranophosphates; achiral phosphate derivatives, such as 3'-0'-5'-S-phosphorothioate, 3'-S-5'-0- phosphorothioate, 3'-CH₂-5'-0-phosphonate and 3'-NH-5'-0 phosphoroamidate; and peptide nucleic acids in which the entire ribose phosphodiester backbone is replaced with a peptide linkage. In some embodiments, modifications of the backbone, sugars and/or heterocyclic bases may increase stability and/or binding affinity.

[0031] "Percent (%) sequence identity" with respect to polynucleotide or polypeptide sequences is defined as the percentage of bases or amino acid residues in a candidate sequence that are identical with the bases or amino acid residues in another sequence, after aligning the two sequences. Gaps can be introduced into the sequence alignment, if necessary, to achieve the maximum percent sequence identity. Conservative substitutions are not considered as part of the sequence identity. Alignment for purposes of determining percent (%) sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer methods and programs such as BLAST, BLAST-2, ALIGN, FASTA (available in the Genetics Computing Group (GCG) package, from Madison, Wisconsin, USA), or Megalign (DNASTAR). Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0032] For instance, percent (%) amino acid sequence identity values may be obtained by using the WU-BLAST-2 computer program described in Altschul et al., Methods in Enzymology, 1996, 266:460-480. Many search parameters in the WU- BLAST-2 computer program can be adjusted by those skilled in the art. For example, some of the adjustable parameters can be set with the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = 11, and scoring matrix = BLOSUM62. When WU-BLAST-2 is used, a % amino acid sequence identity value is determined by dividing (a) the number of matching identical amino acid residues between the amino acid sequence of a first protein of interest and the amino acid sequence of a second protein of interest as determined by WU-BLAST-2 by (b) the total number of amino acid residues of the first protein of interest.

[0033] Percent amino acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 described in Altschul et al., Nucleic Acids Res., 1997, 25:3389-3402. The NCBI-BLAST2 sequence comparison program may be downloaded from http://www.ncbi.nlm.nih.gov or otherwise obtained from the National Institute of Health, Bethesda, MD. NCBI-BLAST2 uses several adjustable search parameters. The default values for some of those adjustable search parameters are, for example, unmask = yes, strand = all, expected occurrences = 10, minimum low complexity length = 15/5, multi-pass e-value = 0.01, constant for multi-pass = 25, dropoff for final gapped alignment = 25 and scoring matrix = BLOSUM62.

[0034] In situations where NCBI-BLAST2 is used for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:

[0035] 100 times the fraction X/Y

[0036] where X is the number of amino acid residues scored as identical matches by the sequence alignment program NCBI-BLAST2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the

% amino acid sequence identity of B to A.

Hyper glycosylated interferon variants of a parent interferon

[0037] Some embodiments disclosed herein relate to a hyperglycosylated interferon variant of a parent interferon that is the parent interferon that has been modified to include a peptide extension inserted at a terminal region, where the peptide extension includes one or more glycosylation sites.

Peptide Extension

[0038] As used herein, the term "peptide extension" refers to any polymer of consecutive amino acid residues. A peptide extension can be of various lengths, for example, ranging from a single amino acid residue to a peptide of 500 amino acid residues. In some embodiments, the peptide extension can be at least about 4 amino acids in length, alternatively at least about 10 amino acids in length, alternatively at least about 20 amino acids in length, alternatively at least about 30 amino acids in length, alternatively at least about 40 amino acids in length, alternatively at least about 50 amino acids in length, alternatively at least about 60 amino acids in length, alternatively at least about 70 amino acids in length, alternatively at least about 80 amino acids in length, alternatively at least about 90 amino acids in length, alternatively at least about 100 amino acids in length, alternatively at least about 110 amino acids in length, alternatively at least about 120 amino acids in length, alternatively at least about 130 amino acids in length, alternatively at least about 140 amino acids in length, alternatively at least about 145 amino acids in length, alternatively at least about 150 amino acids in length, alternatively at least about 155 amino acids in length, alternatively at least about 160 amino acids in length, alternatively at least about 165 amino acids in length, alternatively at least about 170 amino acids in length, alternatively at least about 180 amino acids in length, alternatively at least about 190 amino acids in length, alternatively at least about 200 amino acids in length, alternatively at least about 210 amino acids in length, alternatively at least about 220 amino acids in length, alternatively at least about 230 amino acids in length, alternatively at least about 240 amino acids in length, alternatively at least about 250 amino acids in length, alternatively at least about 260 amino acids in length, alternatively at least about 270 amino acids in length, alternatively at least about 280 amino acids in length, alternatively at least about 290 amino acids in length, alternatively at least about 300 amino acids in length, alternatively at least about 310 amino acids in length, alternatively at least about 320 amino acids in length, alternatively at least about 330 amino acids in length, alternatively at least about 340 amino acids in length, alternatively at least about 350 amino acids in length, alternatively at least about 360 amino acids in length, alternatively at least about 370 amino acids in length, alternatively at least about 380 amino acids in length, alternatively at least about 390 amino acids in length, alternatively at least about 400 amino acids in length, alternatively at least about 450 amino acids in length, alternatively at least about 500 amino acids in length, or more.

[0039] The peptide extension can include any number of glycosylation sites. In some embodiments, the peptide extension can include one glycosylation site. In other embodiments, the peptide extension can include two, three or four glycosylation sites. In still other embodiments, the peptide extension can include five, six, seven, eight, nine, or ten glycosylation sites. In yet other embodiments, the peptide extension can include eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty glycosylation sites. In yet still other embodiments, the peptide extension can include twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty glycosylation sites. In yet still other embodiments, the peptide extension can include thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, thirty-eight, thirty-nine, forty, or more glycosylation sites. Each glycosylation site that is included in the peptide extension may be an N-linked glycosylation site or an O-linked glycosylation site.

[0040] In some embodiments, the peptide extension can include at least one glycosylation site. In other embodiments, the peptide extension can include at least two, at least three or at least four glycosylation sites. In still other embodiments, the peptide extension can include at least five, at least six, at least seven, at least eight, at least nine, or at least ten glycosylation sites. In yet other embodiments, the peptide extension can include at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, or at least twenty glycosylation sites. In yet still other embodiments, the peptide extension can include at least twenty-one, at least twenty-two, at least twenty-three, at least twenty-four, at least twenty-five, at least twenty-six, at least twenty-seven, at least twenty-eight, at least twenty-nine, at least thirty glycosylation sites. In yet still other embodiments, the peptide extension can include at least thirty-one, at least thirty-two, at least thirty-three, at least thirty-four, at least thirty-five, at least thirty-six, at least thirty-seven, at least thirty-eight, at least thirty-nine, at least forty, or more glycosylation sites.

[0041] A glycosylation site that is included in the peptide extension may be a non-native glycosylation site that is present in the parent interferon or a non-native glycosylation site that is not present in the parent interferon. For example, when the parent interferon is interferon ocl4, the glycosylation site N-L-S at amino acid positions 31, 32 and 33 of interferon ocl4 can be introduced to the peptide extension inserted in the parent interferon ocl4 as a non-native glycosylation site. As another example, when the parent interferon is interferon β, the glycosylation site N-L-S at amino acid positions 31, 32 and 33 of interferon ocl4 can be introduced to the peptide extension inserted in the parent interferon β as a non-native glycosylation site.

[0042] The peptide extension can be inserted at an amino-terminal and/or a carboxy-terminal region of the parent interferon. In some embodiments, the peptide can be inserted at an amino-terminal region of the parent interferon. In other embodiments, the peptide extension can be inserted at a carboxy-terminal region of the parent interferon. In still other embodiments, the peptide extension can be inserted at an amino-terminal region and a carboxy-terminal region of the parent interferon.

[0043] The terminal region of the parent interferon at which the peptide extension is inserted can be of various lengths. In some embodiments, the terminal region where the peptide extension is inserted at can be an amino-terminal region that consists of the first 15 amino acids at the amino terminus of the parent interferon that excludes any signal peptide in the parent interferon. In other embodiments, the terminal region where the peptide extension is inserted at can be an amino terminal region that consists of the first 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid(s) at the amino terminus of the parent interferon that excludes any signal peptide in the parent interferon. In still other embodiments, the peptide extension can be inserted before the first amino acid at the amino terminus of the parent interferon that excludes any signal peptide in the parent interferon. In still other embodiments, the peptide extension is inserted after the fifteenth amino acid at the amino terminus of the parent interferon that excludes any signal peptide in the parent interferon.

[0044] For example, when the parent interferon is an interferon a, the peptide extension can be inserted between the 29th and the 30th amino acids of the parent interferon , where the positions of the amino acid substitutions are with reference to the sequence alignment numbering set forth in Figure 1. In addition, the peptide extension can also be inserted between the 30th and the 31st amino acids, between the 31st and the 32nd amino acids, between the 32nd and the 33rd amino acids, between the 33rd and the 34th amino acids, between the 34th and the 35th amino acids, between the 35th and the 36th amino acids, between the 36th and the 37th amino acids, between the 37th and the 38th amino acids, between the 38th and the 39th amino acids, between the 39th and the 40th amino acids, between the 41st and the 42nd amino acids, between the 42nd and the 43rd amino acids, between the 43rd and the 44th amino acids, between the 44th and the 45th amino acids, or between the 45th and the 46th amino acids of the parent interferon a, where the positions of the amino acid substitutions are with reference to the sequence alignment numbering set forth in Figure 1.

[0045] As another example, when the parent interferon is interferon β, the peptide extension can be located between the 27th and 28th amino acids of the parent interferon β, wherein the positions of the amino acid substitutions are with reference to the sequence alignment numbering set forth in Figure 1. In addition, the peptide extension can also be inserted between the 28th and 29th amino acids, between the 29th and 30th amino acids, between the 30th and the 31st amino acids, between the 31st and the 32nd amino acids, between the 32nd and the 33rd amino acids, between the 33rd and the 34th amino acids, between the 34th and the 35th amino acids, between the 35th and the 36th amino acids, between the 36th and the 37th amino acids, between the 37th and the 38th amino acids, between the 38th and the 39th amino acids, between the 39th and the 40th amino acids, between the 41st and the 42nd amino acids, between the 42nd and the 43rd amino acids, or between the 43rd and the 44th amino acids of the parent interferon β, where the positions of the amino acid substitutions are with reference to the sequence alignment numbering set forth in Figure 1.

[0046] As still another example, when the parent interferon is interferon γ, the peptide extension can be located between the 23rd and 24th amino acids of the parent interferon γ. In addition, the peptide extension can also be inserted between the 24th and the 25th amino acids, between the 25th and the 26th amino acids, between the 26th and the 27th amino acids, between the 27th and the 28th amino acids, between the 28th and the 29th amino acids, between the 29th and the 30th amino acids, between the 30th and the 31st amino acids, between the 31st and the 32nd amino acids, between the 32nd and the 33rd amino acids, between the 33rd and the 34th amino acids, between the 34th and the 35th amino acids, between the 35th and the 36th amino acids, between the 36th and the 37th amino acids, between the 37th and the 38th amino acids, between the 38th and the 39th amino acids, or between the 39th and the 40th amino acids of the parent interferon γ.

[0047] As yet still another example, when the parent interferon is interferon λΐ, the peptide extension can be located between the 19th and 20th amino acids of the parent interferon λΐ. In addition, the peptide extension can also be inserted between the 20th and 21st amino acids, between the 21st and 22nd amino acids, between the 22nd and 23rd amino acids, between the 23rd and the 24th amino acids, between the 24th and the 25th amino acids, between the 25th and the 26th amino acids, between the 26th and the 27th amino acids, between the 27th and the 28th amino acids, between the 28th and the 29th amino acids, between the 29th and the 30th amino acids, between the 30th and the 31st amino acids, between the 31st and the 32nd amino acids, between the 32nd and the 33rd amino acids, between the 33rd and the 34th amino acids, or between the 34th and the 35th amino acids of the parent interferon λΐ.

[0048] As another example, when the parent interferon is interferon λ2, the peptide extension can be located between the 25th and 26th amino acids of the parent interferon λ2. In addition, the peptide extension can also be inserted between the 26th and 27th amino acids, between the 27th and 28th amino acids, between the 28th and 29th amino acids, between the 29th and 30th amino acids, between the 30th and the 31st amino acids, between the 31st and the 32nd amino acids, between the 32nd and the 33rd amino acids, between the 33rd and the 34th amino acids, between the 34th and the 35th amino acids, between the 35th and the 36th amino acids, between the 36th and the 37th amino acids, between the 37th and the 38th amino acids, between the 38th and the 39th amino acids, between the 39th and the 40th amino acids, between the 41st and the 42nd amino acids, or between the 42nd and the 43rd amino acids of the parent interferon λ2.

[0049] As yet still another example, when the parent interferon is interferon λ3, the peptide extension can be located between the 21st and 22nd amino acids of the parent interferon λ3. In addition, the peptide extension can also be inserted between the 22nd and 23rd amino acids, between the 23rd and the 24th amino acids, between the 24th and the 25th amino acids, between the 25th and the 26th amino acids, between the 26th and the 27th amino acids, between the 27th and the 28th amino acids, between the 28th and the 29th amino acids, between the 29th and the 30th amino acids, between the 30th and the 31st amino acids, between the 31st and the 32nd amino acids, between the 32nd and the 33rd amino acids, between the 33rd and the 34th amino acids, between the 34th and the 35th amino acids, between the 35th and the 36th amino acids, or between the 36th and the 37th amino acids of the parent interferon λ3.

[0050] In some embodiments, the terminal region where the peptide extension is inserted at can be a carboxy-terminal region that consists of the last 15 amino acids at the carboxy-terminus of the parent interferon. In other embodiments, the terminal region where the peptide extension is inserted at can be a carboxy-terminal region that consists of the last 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid(s) at the carboxy- terminus of the parent interferon. In some embodiments, the peptide extension can be inserted after the last amino acid at the carboxy-terminus of the parent interferon. In other embodiments, the peptide extension is not inserted after the last amino acid at the carboxy-terminus of the parent interferon. In still other embodiments, the peptide extension includes at least two glycosylation sites and is not inserted after the last amino acid at the carboxy-terminus of the parent interferon. In yet other embodiments, the parent interferon can be interferon β, where the peptide extension is not inserted after the last amino acid at the carboxy-terminus of the parent interferon. In yet still other embodiments, the parent interferon can be interferon β, where the peptide extension includes at least two glycosylation sites and is not inserted after the last amino acid at the carboxy-terminus of the parent interferon.

Parent interferon

[0051] As used herein, a "parent interferon" can be any interferon, including naturally-occurring and non-naturally occurring interferons. Examples of interferons that can be the parent interferon include, but are not limited to, Type I interferons, Type Π interferons, or Type ΙΠ interferons.

[0052] As used herein, a "naturally occurring interferon" refers to an interferon that can be found in nature as distinct from being artificially produced by man. For example, an interferon that is produced by an organism in nature, where the organism has not been intentionally modified by man is naturally occurring. Examples of organism that can produce naturally occurring interferons include, but are not limited to, non- mammalian vertebrates, such as birds (for example, turkey, chicken, and duck) and fish (for example, zebrafish); and mammalian vertebrate, such as human, pig, mouse, dog, cat, horse, rat, cattle and sheep.

[0053] A naturally occurring interferon can be at least about 50 amino acids in length, at least about 100 amino acids in length, alternatively about 110 amino acids in length, alternatively about 120 amino acids in length, alternatively about 130 amino acids in length, alternatively about 140 amino acids in length, alternatively about 150 amino acids in length, alternatively about 160 amino acids in length, alternatively about 170 amino acids in length, alternatively about 180 amino acids in length, alternatively about 190 amino acids in length, alternatively about 200 amino acids in length, alternatively about 210 amino acids in length, alternatively about 220 amino acids in length, alternatively about 230 amino acids in length, alternatively about 240 amino acids in length, alternatively about 250 amino acids in length, alternatively about 260 amino acids in length, alternatively about 270 amino acids in length, alternatively about 280 amino acids in length, alternatively about 290 amino acids in length, alternatively about 300 amino acids in length, alternatively about 310 amino acids in length, alternatively about 320 amino acids in length, alternatively about 330 amino acids in length, alternatively about 340 amino acids in length, alternatively about 350 amino acids in length, alternatively about 360 amino acids in length, alternatively about 370 amino acids in length, alternatively about 380 amino acids in length, alternatively about 390 amino acids in length, alternatively about 400 amino acids in length, alternatively about 410 amino acids in length, alternatively about 420 amino acids in length, alternatively about 430 amino acids in length, alternatively about 440 amino acids in length, alternatively about 450 amino acids in length, alternatively about 460 amino acids in length, alternatively about 470 amino acids in length, alternatively about 480 amino acids in length, alternatively about 490 amino acids in length, alternatively about 500 amino acids in length, or more. In some embodiments, the parent interferon can be a naturally-occurring interferon.

[0054] As used herein, the term "non-naturally occurring interferon" is used interchangeably with the term "synthetic interferon," and refers to an interferon that cannot be found in nature and is artificially produced by man. Non-naturally occurring interferons include, but are not limited to, hybrid interferons, consensus interferons, fusion interferons, recombinant interferons, and other non-naturally occurring variants of a naturally-occurring or a non-naturally occurring interferon. [0055] In some embodiments, the parent interferon can be a non-naturally occurring interferon. In an embodiment, the parent interferon can be a hybrid interferon. In another embodiment, the parent interferon can be a consensus interferon. In still another embodiment, the parent interferon can be a fusion interferon. In yet another embodiment, the parent interferon can be a recombinant interferon.

[0056] A non-naturally occurring interferon can be of various lengths. In some embodiments, a non-naturally occurring interferon is at least about 50 amino acids in length, alternatively at least about 60 amino acids in length, alternatively at least about 70 amino acids in length, alternatively at least about 80 amino acids in length, alternatively at least about 90 amino acids in length, alternatively at least about 100 amino acids in length, alternatively at least about 110 amino acids in length, alternatively at least about 120 amino acids in length, alternatively at least about 130 amino acids in length, alternatively at least about 140 amino acids in length, alternatively at least about 145 amino acids in length, alternatively at least about 150 amino acids in length, alternatively at least about 155 amino acids in length, alternatively at least about 160 amino acids in length, alternatively at least about 165 amino acids in length, alternatively at least about 170 amino acids in length, alternatively at least about 180 amino acids in length, alternatively at least about 190 amino acids in length, alternatively at least about 200 amino acids in length, alternatively at least about 210 amino acids in length, alternatively at least about 220 amino acids in length, alternatively at least about 230 amino acids in length, alternatively at least about 240 amino acids in length, alternatively at least about 250 amino acids in length, alternatively at least about 260 amino acids in length, alternatively at least about 270 amino acids in length, alternatively at least about 280 amino acids in length, alternatively at least about 290 amino acids in length, alternatively at least about 300 amino acids in length, alternatively at least about 310 amino acids in length, alternatively at least about 320 amino acids in length, alternatively at least about 330 amino acids in length, alternatively at least about 340 amino acids in length, alternatively at least about 350 amino acids in length, alternatively at least about 360 amino acids in length, alternatively at least about 370 amino acids in length, alternatively at least about 380 amino acids in length, alternatively at least about 390 amino acids in length, alternatively at least about 400 amino acids in length, alternatively at least about 410 amino acids in length, alternatively at least about 420 amino acids in length, alternatively at least about 430 amino acids in length, alternatively at least about 440 amino acids in length, alternatively at least about 450 amino acids in length, alternatively at least about 460 amino acids in length, alternatively at least about 470 amino acids in length, alternatively at least about 480 amino acids in length, alternatively at least about 490 amino acids in length, alternatively at least about 500 amino acids in length, alternatively at least about 550 amino acids in length, alternatively at least about 600 amino acids in length, alternatively at least about 650 amino acids in length, alternatively at least about 700 amino acids in length, alternatively at least about 750 amino acids in length, alternatively at least about 800 amino acids in length, alternatively at least about 850 amino acids in length, alternatively at least about 900 amino acids in length, alternatively at least about 950 amino acids in length, alternatively at least about 1000 amino acids in length, or more.

Hybrid interferon

[0057] As used herein, a "hybrid interferon" is a hybrid polypeptide having an amino acid sequence comprising discrete sub- sequences corresponding in amino acid identity and number to sub- sequences of more than one naturally-occurring and/or non- naturally occurring interferons. Each of the naturally-occurring and/or non-naturally occurring interferons from which the discrete sub-sequences are derived from may be the same or different from each other. For example, a hybrid interferon can include discrete sub- sequences from two naturally-occurring and/or non-naturally interferons, where the two naturally-occurring and/or non-naturally interferons are different from each other. As another example, a hybrid interferon can include discrete sub- sequences from three, four, five, six, seven, eight, nine, or ten naturally-occurring and/or non-naturally occurring interferons.

[0058] For example, the discrete sub- sequences can be selected from naturally-occurring IFN-a2b, naturally-occurring IFN-al4, naturally-occurring IFN-βΙ, and naturally-occurring IFN-ω, and the amino acid sequence of the hybrid interferon differs from each of the amino acid sequences of naturally occurring IFN-a2b, naturally- occurring IFN-al4, naturally-occurring IFN-βΙ, and naturally-occurring IFN-co, respectively. As another example, the discrete sub- sequences can be selected from naturally-occurring IFN-a2b, naturally-occurring IFN-al4, naturally-occurring IFN-βΙ, Infergen® consensus IFN-a, and naturally-occurring IFN-ω, and the amino acid sequence of the hybrid interferon differs from each of the amino acid sequences of naturally- occurring IFN-a2b, naturally-occurring IFN-al4, naturally-occurring IFN-βΙ, Infergen® consensus IFN-α, and naturally-occurring IFN-ω, respectively. As still another example, the discrete sub-sequences can be selected from naturally-occurring IFN-λΙ, naturally- occurring ΙΡΝ-λ2, and naturally occurring ΙΡΝ-λ3, and the amino acid sequence of the hybrid interferon differs from each of the amino acid sequences of naturally-occurring IFN-λΙ, naturally-occurring IFN- 2, and naturally occurring ΙΡΝ-λ3, respectively.

[0059] In some embodiments, a hybrid interferon can include, in the order from N-terminus to C-terminus, from about 2 to about 90, for example, from about 2 to about 5, from about 5 to about 7, from about 7 to about 10, from about 10 to about 15, from about 15 to about 20, from about 20 to about 25, from about 25 to about 30, from about 30 to about 35, from about 35 to about 40, from about 40 to about 45, from about 45 to about 50, from about 50 to about 55, from about 55 to about 60, from about 60 to about 65, from about 65 to about 70, from about 75 to about 80, from about 80 to about 85, or from about 85 to about 90 contiguous amino acids of two, three, or four interferons selected from human IFN-a2b (SEQ ID NO: 81), human IFN-al4 (SEQ ID NO: 90), human IFN-βΙ (SEQ ID NO: 97), and IFN-COl (SEQ ID NO: 99).

Consensus interferon

[0060] As used herein, a "consensus interferon" is an interferon having a consensus amino acid sequence that is derived by aligning the amino acid sequences of three or more naturally occurring and/or non-naturally occurring interferons, and identifying the amino acids that are shared by at least two of the naturally occurring and/or non-naturally occurring interferon sequences. For example, a consensus interferon can include a consensus amino acid sequence that is derived by aligning the amino acid sequences of three, four, five, six, seven, eight, nine, or ten naturally occurring and/or non-naturally occurring interferons, and identifying amino acids that are shared by at least two, three, five, six, seven, eight, nine, or ten of the naturally occurring and/or non- naturally occurring interferon sequences.

[0061] As an example, a consensus interferon can be a sequence derived from aligning the sequences of naturally occurring human IFN-a2b, naturally-occurring human IFN-al4, and naturally-occurring human IFN-βΙ. As another example, a consensus interferon can be a sequence derived from aligning the sequences of naturally occurring human IFN-a2b, naturally-occurring human IFN-al4, and naturally-occurring human IFN-col . As still another example, a consensus interferon can be a sequence derived from aligning the sequences of naturally occurring human IFN-a2b, naturally-occurring human IFN-βΙ, and naturally-occurring human IFN-col. As yet another example, a consensus interferon can be a sequence derived from aligning the sequences of naturally occurring human IFN-al4, naturally-occurring human IFN-βΙ, and naturally-occurring human IFN- col. As still yet another example, a consensus interferon can be a sequence derived from aligning the sequences of naturally occurring human IFN-a2b, naturally-occurring human IFN-al4, naturally-occurring human IFN-βΙ, and naturally-occurring human IFN-col. As still yet another example, a consensus interferon can be a sequence derived from aligning the sequences of naturally-occurring human IFN-λΙ, naturally-occurring human ΙΡΝ-λ2, and naturally-occurring human ΙΡΝ-λ3.

[0062] An example of consensus interferon is Infergen® consensus IFN-a (Three Rivers Pharmaceuticals, Warrendale, PA). In some embodiments, a consensus sequence can be derived by including one or more consensus interferons, such as Infergen® consensus IFN-a, in the amino acid sequence alignment.

Fusion interferon

[0063] As used herein, a "fusion interferon" is an interferon comprising a fusion partner, such as one or more heterologous peptides or polypeptides. Suitable fusion partners include, but are not limited to, peptides and polypeptides that confer enhanced stability in vivo (for example, enhanced serum half-life); peptides and polypeptides that can provide ease of purification (for example, (His)_n) and the like; peptides and polypeptides that can provide for secretion of the fusion protein from a cell, and the like; peptides and polypeptides that can provide an epitope tag, such as GST, hemagglutinin (for example, CYPYDVPDYA), FLAG (for example, DYKDDDDK), c- myc (for example, CEQKLISEEDL), and the like; peptides and polypeptides that can provide a detectable signal, such as an enzyme that generates a detectable product (for example, β-galactosidase and luciferase), and the like; a peptide or polypeptide that is itself detectable (for example, a green fluorescent protein) and the like; and peptides and polypeptides that can provide for multimerization, such as a multimerization domain (for example, an Fc portion of an immunoglobulin) and the like.

[0064] Various signal peptide sequences can be incorporated into an interferon as a fusion partner to provide for secretion of the fusion protein from a cell. In some embodiments, the fusion partner can be a signal peptide that provides for secretion from a mammalian cell. Such signal peptides include, but are not limited to, a signal peptide from IFN-β and a signal peptide from IFN-al4. Methods of producing a fusion interferon comprising an IFN-al4 or an IFN-β signal peptide are described in U.S. Patent Publication No. 2006-0204473, the contents of which are hereby incorporated by reference in its entirety. In other embodiments, the fusion partner can be a bacterial secretion signal peptide. Such signal peptides include, but are not limited to, the secretion signal of Braun's lipoprotein of E. coli, S. marcescens, E. amylosora, M. morganii, and P. mimbilis; the TraT protein of E. coli and Salmonella; the penicillinase (PenP) protein of B. licheniformis, B. cereus and S. aureus; pullulanase proteins of Klebsiella pneumoniae and Klebsiella aerogenese; E. coli lipoproteins lpp-28, Pal, RplA, RplB, OsmB, NIpB, and Orll7; chitobiase protein of V. harseyi; the P-l,4-endoglucanase protein of Pseudomonas solanacearum; the Pal and Pep proteins of H. influenzae; the Oprl protein of P. aeruginosa; the MalX and AmiA proteins of S. pneumoniae; the 34 kda antigen and TpmA protein of Treponema pallidum; the P37 protein of Mycoplasma hyorhinis; the neutral protease of Bacillus amyloliquefaciens; and the 17 kda antigen of Rickettsia rickettsii. In still other embodiments, the fusion partner can be a yeast secretion signal peptide. Secretion signal peptides that can be used in yeast are known in the art. See, for example, U.S. Patent No. 5,712,113, the contents of which are hereby incorporated by reference in its entirety. Examples of signal peptides include, but are not limited to, the endogenous signal peptide for interferons, including the signal peptide of type I, II and ΙΠ interferons; and the endogenous signal pepide for any known cytokine, such as the signal peptide of erythropoietin (EPO), TGF-βΙ, TNF, ILl-oc, and ILl-β. Nucleotide sequences of the non-limiting examples of signal peptides are given in SEQ ID NOs: 121-128. For example, SEQ ID NO: 121 shows the sequence of signal peptide of human insulin, SEQ ID NO: 122 shows the sequence of an artificial signal peptide, SEQ ID NO: 123 shows the sequence of signal peptide of human CD33, SEQ ID NO: 124 shows the sequence of signal peptide of human EPO, SEQ ID NO: 125 shows the sequence of signal peptide of human G-CSF, SEQ ID NO: 126 shows the sequence of signal peptide of human growth hormone 1, SEQ ID NO: 127 shows the sequence of signal peptide of human interferon beta, and SEQ ID NO: 128 shows the sequence of signal peptide of human insulin like growth factor 1A.

[0065] In some embodiments, a fusion interferon can further include a protease cleavage site that is positioned between the fusion partner and the remainder of the fusion interferon. Examples of proteolytic cleavage sites that can be included in a fusion interferon disclosed herein include, but are no limited to, an enterokinase cleavage site: (Asp)₄Lys; a factor Xa cleavage site: Ile-Glu-Gly-Arg; a thrombin cleavage site, such as Leu-Val-Pro-Arg-Gly-Ser; a renin cleavage site, such as His-Pro-Phe-His-Leu-Val-Ile- His; a collagenase cleavage site, such as X-Gly-Pro (where X is any amino acid); a trypsin cleavage site, such as Arg-Lys; a viral protease cleavage site, such as a viral 2A or 3C protease cleavage site, including, but not limited to, a protease 2A cleavage site from a picornavirus described in Sommergruber et al., Virol., 1994, 198:741-745, a Hepatitis A virus 3C cleavage site described in Schultheiss et al., J. Virol., 1995, 69:1727-1733, human rhinovirus 2A protease cleavage site described in Wang et al., Biochem. Biophys. Res. Comm., 1997, 235:562-566, and a picornavirus 3 protease cleavage site described in Walker et al., Biotechnol., 1994, 12:601-605.

Recombinant interferon

[0066] As used herein, a "recombinant interferon" is a non-naturally occurring interferon that is produced utilizing recombinant techniques. For example, a recombinant interferon can be produced by genetic engineering techniques known in the art, such as recursive sequence recombination of nucleic acid segments, diversity generation methods (such as shuffling) of nucleotides, or manipulation of isolated segments of polynucleotides or polypeptides. Various recombinant interferons are known in the art, including recombinant interferon alpha-2b such as Intron-A^® interferon available from Schering Corporation, Kenil worth, NJ; recombinant interferon alpha-2a such as Roferon- A^® interferon available from Hoffmann-La Roche, Nutley, NJ; recombinant interferon alpha- 2C such as Berofor alpha 2 interferon available from Boehringer Ingelheim Pharmaceutical, Inc., Ridgefield, CT; interferon alpha-nl, a purified blend of natural alpha interferons, available from the Sumitomo Pharmaceuticals Co., Japan, under the trade name of Sumiferon^® or from the Glaxo- Wellcome Ltd., London, Great Britain under the trade name of Wellferon^®; interferon alpha-n3, a mixture of natural alpha interferons made by Interferon Sciences and available from the Purdue Frederick Co., Norwalk, CT, under the trade name of Alferon^®; and recombinant interferon γ such as interferon gamma IB available from InterMune under the trade name of Actimmune^®.

[0067] In some embodiments, the parent interferon can include one or more modified amino acids. Examples of modified amino acid include, but are not limited to, a glycosylated amino acid, a PEGylated amino acid, a farnesylated amino acid, a carboxylated amino acid, a phosphorylated amino acid (for example, phosphotyrosine, phosphoserine, and phospho threonine), an acetylated amino acid, a biotinylated amino acid, an amino acid conjugated to a lipid moiety, an amino acid conjugated to an organic derivatizing agent, and the like.

[0068] In some embodiments, the parent interferon can be a naturally occurring or non-naturally occurring interferon that includes one or more polyethylene glycol (PEG) moieties. The PEG molecule(s) can be conjugated to one or more amino acid side chains of the PEGlyated interferon. The PEG can be coupled directly to an interferon without a linking group, or through an amino group, a sulfhydryl group, a hydroxyl group, or a carboxyl group. Methods for attaching a PEG to a polypeptide are known in the art, and any known method can be used. See, for example, Park et al, Anticancer Res., 1981, 1:373-376; and U.S. Patent No. 5,985,265. In some embodiments, the PEGylated interferon can contain a PEG moiety on only one amino acid. In other embodiments, a PEGylated interferon contains two, three, four, five, six, seven, eight, nine or ten PEG moieties on two, three, four, five, six, seven, eight, nine or ten different amino acid residues. In some embodiments, the parent interferon can be a PEGylated interferon that is PEGylated at or near the amino-terminus, where a peptide extension can be inserted at the carboxy-terminal region of the parent interferon. For example, the PEG moiety can be conjugated to the interferon at one or more amino acid residues from amino acid 1 through amino acid 4, or from amino acid 5 through amino acid 10. In other embodiments, the parent interferon can be a PEGylated interferon that is PEGylated at or near the carboxy-terminus, where a peptide extension can be inserted at the amino- terminal region of the parent interferon. In an embodiment, the PEGylated interferon is PEGylated at one or more amino acid residues at one or more residues from amino acid 100 through amino acid 114. In some embodiments, the addition of one or more pegylation sites can provide PEG-derivatized interferon with reduced serum clearance.

[0069] In some embodiments, the PEG can be a monomethoxyPEG molecule (mPEG) that reacts with a primary amine group on the subject polypeptide. Methods of modifying polypeptides with monomethoxy PEG via reductive alkylation are known in the art. See, for example, Chamow et al., Bioconj. Chem., 1994, 5:133-140.

[0070] As discussed herein, various interferons can be a parent interferon. A non-limiting list of examples of interferons that can be the parent interferon includes, but is not limited to, naturally occurring and non-naturally occurring Type I interferons, naturally occurring and non-naturally occurring Type Π interferons, and naturally occurring and non-naturally occurring Type ΠΙ interferons.

Type I interferons

[0071] As discussed herein, various Type I interferons can be a parent interferon. A non-limiting list of examples of Type I interferons that can be a parent interferon includes, but is not limited to, naturally-occurring and non-naturally occurring interferon a (IFN a), naturally-occurring and non-naturally occurring interferon β (IFN β), naturally-occurring and non-naturally occurring interferon κ (IFN κ), naturally-occurring and non-naturally occurring interferon ω (IFN ω), and naturally-occurring and non- naturally occurring interferon τ (IFN τ).

[0072] In some embodiments, the parent interferon can be a naturally occurring or non-naturally-occurring interferon a. In other embodiments, the parent interferon can be a naturally-occurring or non-naturally occurring interferon β. In still other embodiments, the parent interferon can be a naturally-occurring or non-naturally occurring interferon τ. In yet other embodiments, the parent interferon can be a naturally- occurring or non-naturally occurring interferon CO. In still yet other embodiments, the parent interferon can be a naturally-occurring or non-naturally occurring interferon κ.

[0073] Examples of interferon a include, but are not limited to, interferon alfacon-1, naturally-occurring and non-naturally occurring interferon al (IFN al), naturally-occurring and non-naturally occurring interferon a2a (IFN a2a), naturally- occurring and non-naturally occurring interferon a2b (IFN a2b), naturally-occurring and non-naturally occurring interferon a4a (IFN a4a), naturally-occurring and non-naturally occurring interferon a4b (IFN a4b), naturally-occurring and non-naturally occurring interferon a5 (IFN a5), naturally-occurring and non-naturally occurring interferon a6 (IFN a6), naturally-occurring and non-naturally occurring interferon a7 (IFN a7), naturally- occurring and non-naturally occurring interferon a8 (IFN a8), naturally-occurring and non-naturally occurring interferon alO (IFN alO), naturally-occurring and non-naturally occurring interferon al3 (IFN al3), naturally-occurring and non-naturally occurring interferon al4 (IFN al4), naturally-occurring and non-naturally occurring interferon al6 (IFN al6), naturally-occurring and non-naturally occurring interferon al7 (IFN al7), naturally-occurring and non-naturally occurring interferon a21 (IFN a21), naturally- occurring and non-naturally occurring interferon aH (IFN aH), naturally-occurring and non-naturally occurring interferon al (IFN al), naturally-occurring and non-naturally occurring interferon aJl (IFN aJl), naturally-occurring and non-naturally occurring interferon a2c (IFN-a2c), naturally-occurring and non-naturally occurring interferon ad (IFN-ad), IFN-con2, and IFN-con3. Other non-limiting examples of interferon a include IFN- a described in U.S. Patent No. 4,695,623.

[0074] In an embodiment, the parent interferon can be interferon alfacon-1. In another embodiment, the parent interferon can be interferon alpha-2a (IFN 0c2a). In yet another embodiment, the parent interferon can be interferon alpha-2b (IFN 0c2b). In still another embodiment, the parent interferon can be interferon alpha-1 (IFN al). In yet still another embodiment, the parent interferon can be interferon alpha- 14 (IFN 0cl4).

[0075] Examples of interferon β include, but are not limited to, naturally- occurring IFN-β; IFN-pia, for example, Avonex® (Biogen, Inc.), and Rebif® (Merck Serono, SA); IFN-pib (Betaseron®; Berlex); and the like. Amino acid sequences of IFN- β are publicly available; for example, human IFN-βΙ amino acid sequence is available under GenBank Accession No. NP_002167 (SEQ ID NO: 97).

[0076] Examples of interferon τ include, but are not limited to, naturally- occurring IFN-tau; Tauferon® (Pepgen Corp.); and the like. IFN-tau may comprise an amino acid sequence set forth in any one of GenBank Accession Nos. P15696; P56828; P56832; P56829; P56831; Q29429; Q28595; Q28594; Q08071; Q08070; Q08053; P56830; P28169; P28172; and P28171.

[0077] Examples of interferon ω include, but are not limited to, naturally- occurring IFN-co; non-naturally occurring IFN-CO, such as recombinant IFN-co; and the like. IFN-co may comprise an amino acid sequence set forth in any one of GenBank Accession Nos. NP_002168 and AAA70091.

[0078] Examples of interferon κ include, but are not limited to, naturally- occurring IFN-K, non-naturally occurring IFN-κ, and the like. IFN-κ may comprise an amino acid sequence set forth in GenBank Accession No. Q9P0W0.1

Sequence Homology of Type I interferons

[0079] Type I interferons, including interferon α, β, co, τ, and κ, are a family of well-known biomolecules that share a high degree of sequence homology. Conservation in sequence, function, and structure for Type I interferons are described in the art. For example, Piehler et al., J. Biol. Chem., 2002, 275(51):40425-40433 shows that a high degree of sequence similarity exists between different alpha interferons and also between alpha interferons, beta interferons and omega interferons. Schultz et al., Dev. Comp. Immunol., 2004, 28(5):499-508 further shows the sequence conservation between interferons isolated from biologically diverse organisms.

[0080] Figure 1 depicts an amino acid sequence alignment of the human type I interferon (IFN) and the fusion protein of consensus IFN-aConl with human IFN al4 signal peptide (Infergen w A14 Sig). Using softwares known in the art, such as Lasergene and ClustalW, any Type I interferon of interest can be aligned to, with, or against the Majority Sequence in Figure 1. Then, using the numbering of the ruler below the Majority Sequence in Figure 1, the corresponding amino acid position for each amino acid position in Figure 1 can be determined for any Type I interferon of interest.

[0081] Sequence alignment of Type I interferons in Figure 1 provides the correlation shown below in Table 1. For example, it will be apparent to those skilled in the art that the amino acid position 31 in Figure 1 corresponds to position 25 of the sequence of IFN alfacon-1, and thus D31 of IFN alfacon-1 depicted in Figure 1 corresponds to the Aspartic acid (D) at position 25 of the sequence of IFN alfacon-1. Similarly, L32 of IFN alfacon-1 depicted in Figure 1 corresponds to the Leucine (L) at position 26 of the sequence of IFN alfacon-1; P33 of IFN alfacon-1 depicted in Figure 1 corresponds to the Proline (P) at position 27 of the sequence of IFN alfacon-1; R52 of IFN alfacon-1 depicted in Figure 1 corresponds to the Arginine (A) at position 45 of the sequence of IFN alfacon-1; 154 of IFN alfacon-1 depicted in Figure 1 corresponds to the Isoleucine (I) at position 47 of the sequence of IFN alfacon-1; E72 of IFN alfacon-1 depicted in Figure 1 corresponds to the Glutamic acid (E) at position 65 of the sequence of IFN alfacon-1; D74 corresponds to the Aspartic acid (D) at position 67 of the sequence of IFN alfacon-1; N76 corresponds to the Asparagine (N) at position 69 of the sequence of IFN alfacon-1; F78 of IFN alfacon-1 depicted in Figure 1 corresponds to the Phenylalanine (F) at position 71 of the sequence of IFN alfacon-1; K101 of IFN alfacon-1 depicted in Figure 1 corresponds to the Lysine (K) at position 94 of the sequence of IFN alfacon-1; D102 of IFN alfacon-1 depicted in Figure 1 corresponds to the Aspartic acid (D) at position 95 of the sequence of IFN alfacon-1; S104 of IFN alfacon-1 depicted in Figure 1 corresponds to the Serine (S) at position 97 of the sequence of IFN alfacon-1; D108 of IFN alfacon-1 depicted in Figure 1 corresponds to the Aspartic acid (D) at position 101 of the sequence of IFN alfacon-1; S110 of IFN alfacon-1 depicted in Figure 1 corresponds to the Serine (S) at position 103 of the sequence of IFN alfacon-1; E138 of IFN alfacon-1 depicted in Figure 1 corresponds to the Glutamic acid (E) at position 130 of the sequence of IFN alfacon-1; N144K of IFN alfacon-1 depicted in Figure 1 corresponds to the Asparagine (N) at position 136 of the sequence of IFN alfacon-1; and V145 of IFN alfacon-1 depicted in Figure 1 corresponds to the Valine (V) at position 137 of the sequence of IFN alfacon-1.

[0082] Similarly, Table 1 provides that L32 and L33 of IFN β depicted in Figure 1 correspond, respectively, to the Leucine (L) at position 26 and the Leucine (L) at position 27 of the sequence of IFN β. Further, Table 1 shows that IFN β has no amino acid residue present at position 145, where the positions of the amino acid substitutions are with reference to the sequence alignment numbering set forth in Figure 1.

Table 1. Correlation of amino acid positions in Figure 1 and in Type I interferon sequences

' means that no amino acid residue is present in the specific amino acid position in the indicated interferon.

Type II interferons

[0083] Examples of Type II interferons includes, but are not limited to, naturally occurring and non-naturally occurring interferon gamma (IFN-γ).

[0084] In some embodiments, the parent interferon can be a Type II interferon. In some embodiments, the parent interferon can be an IFN-γ. Examples of interferon γ include, but are not limited to, naturally-occurring IFN γ; non-naturally occurring IFN γ, for example, interferon gamma IB available from InterMune under the trade name of Actimmune^®; and the like. IFN γ may comprise an amino acid sequence as set forth in GenBank Accession No. AAB59534.1.

Type III interferons

[0085] Examples of Type III IFNs includes, but are not limited to, naturally- occurring and non-naturally occurring interferon lambda (IFN-λ). Non-limiting examples of IFN-λ include IFN-λΙ, IFN-A2, and IFN-A3. IFN-λΙ, IFN-A2, and ΙΡΝ-λ3 share significant sequence homology. See Johonston, Proc. Am. Thorac. Soc, 2007, 4:267-270.

[0086] In some embodiments, the parent interferon can be a Type III interferon. In some embodiments, the parent interferon can be an IFN-λ. Example of IFN-λ include, but are not limited to, naturally-occurring and non-naturally occurring IFN lambda 1, naturally- occurring and non-naturally occurring IFN lambda 2, and naturally-occurring and non- naturally occurring IFN lambda 3. For example, interferon lambda 1 may comprise an amino acid sequence as set forth in GenBank Accession No. NP_742152.1, interferon lambda 2 may comprise an amino acid sequence as set forth GenBank Accession No. NP_742150.1, and interferon lambda 3 may comprise an amino acid sequence as set forth in GenBank Accession No. NP_742151.2.

Hyperglycosylated interferon variants

[0087] In some embodiments, a hyperglycosylated interferon variant of a parent interferon can be the parent interferon that has been modified to include a peptide extension inserted at a terminal region, where the peptide extension can include at least one glycosylation site. In some embodiments, the peptide extension can include at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten glycosylation sites. [0088] The hyperglycosylated interferon can be of various lengths. In some embodiments, a hyperglycosylated interferon variant of a parent interferon can be at least about 50 amino acids in length, alternatively at least about 60 amino acids in length, alternatively at least about 70 amino acids in length, alternatively at least about 80 amino acids in length, alternatively at least about 90 amino acids in length, alternatively at least about 100 amino acids in length, alternatively at least about 110 amino acids in length, alternatively at least about 120 amino acids in length, alternatively at least about 130 amino acids in length, alternatively at least about 140 amino acids in length, alternatively at least about 145 amino acids in length, alternatively at least about 150 amino acids in length, alternatively at least about 155 amino acids in length, alternatively at least about 160 amino acids in length, alternatively at least about 165 amino acids in length, alternatively at least about 170 amino acids in length, alternatively at least about 175 amino acids in length, alternatively at least about 180 amino acids in length, alternatively at least about 185 amino acids in length, alternatively at least about 190 amino acids in length, alternatively at least about 195 amino acids in length, alternatively at least about 200 amino acids in length, alternatively at least about 205 amino acids in length, alternatively at least about 210 amino acids in length, alternatively at least about 215 amino acids in length, alternatively at least about 220 amino acids in length, alternatively at least about 230 amino acids in length, alternatively at least about 240 amino acids in length, alternatively at least about 250 amino acids in length, alternatively at least about 260 amino acids in length, alternatively at least about 270 amino acids in length, alternatively at least about 280 amino acids in length, alternatively at least about 290 amino acids in length, alternatively at least about 300 amino acids in length, alternatively at least about 310 amino acids in length, alternatively at least about 320 amino acids in length, alternatively at least about 330 amino acids in length, alternatively at least about 340 amino acids in length, alternatively at least about 350 amino acids in length, alternatively at least about 360 amino acids in length, alternatively at least about 370 amino acids in length, alternatively at least about 380 amino acids in length, alternatively at least about 390 amino acids in length, alternatively at least about 400 amino acids in length, alternatively at least about 410 amino acids in length, alternatively at least about 420 amino acids in length, alternatively at least about 430 amino acids in length, alternatively at least about 440 amino acids in length, alternatively at least about 450 amino acids in length, alternatively at least about 460 amino acids in length, alternatively at least about 470 amino acids in length, alternatively at least about 480 amino acids in length, alternatively at least about 490 amino acids in length, alternatively at least about 500 amino acids in length, alternatively at least about 510 amino acids in length, alternatively at least about 520 amino acids in length, alternatively at least about 530 amino acids in length, alternatively at least about 540 amino acids in length, alternatively at least about 550 amino acids in length, alternatively at least about 560 amino acids in length, alternatively at least about 570 amino acids in length, alternatively at least about 580 amino acids in length, alternatively at least about 590 amino acids in length, alternatively at least about 600 amino acids in length, or more.

[0089] The hyperglycosylated interferon variants disclosed herein can have various molecular weight. In some embodiments, the molecular weight of the hyperglycosylated interferon variants is at least 70 kD, at least 75 kD, at least 80 kD, at least 85 kD, at least 90 kD, at least 95 kD, at least 100 kD, at least 105 kD, at least 110 kD, at least 115 kD, at least 120 kD, at least 125 kD, or at least 130 kD. In some embodiments, the molecular weight of the hyperglycosylated interferon variants is in the range of about 70 kD to about 200 kD. In other embodiments, the molecular weight of the hyperglycosylated interferon variants is in the range of about 70 kD to about 150 kD. In still other embodiments, the molecular weight of the hyperglycosylated interferon variants is in the range of about 70 kD to about 100 kD. In yet still other embodiments, the molecular weight of the hyperglycosylated interferon variants is in the range of about 80 kD to about 100 kD.

[0090] In some embodiments, the parent interferon can be an interferon a and the peptide extension can be located between the 29th and the 30th amino acid residues of the parent interferon a, wherein the positions of the amino acid substitutions are with reference to the sequence alignment numbering set forth in Figure 1. In an embodiment, the parent interferon can be selected from interferon alfacon-1, interferon a2a, interferon a2b, interferon al, interferon a4a, interferon a4b, interferon a5, interferon a7, interferon a8, interferon alO, interferon al3, interferon al4, interferon al6, interferon al7, interferon a21, interferon aH, interferon al, interferon aJl, recombinant interferon alpha-2b, recombinant interferon alpha- 2a, recombinant interferon alpha-2C, interferon alpha-nl, interferon alpha-n3, IFN-con2, or IFN-con3, and the peptide extension can be located between amino acids G29 and C30 of the parent interferon, where the positions of the amino acid substitutions are with reference to the sequence alignment numbering set forth in Figure 1. In another embodiment, the parent interferon can be interferon a6 and the peptide extension can be located between amino acids D29 and C30 of interferon a6, where the positions of the amino acid substitutions are with reference to the sequence alignment numbering set forth in Figure 1.

[0091] In some embodiments, the parent interferon can be interferon β and the peptide extension can be located between the amino acids S27 and M28 of interferon β, wherein the positions of the amino acid substitutions are with reference to the sequence alignment numbering set forth in Figure 1.

[0092] In other embodiments, the parent interferon can be interferon γ and the peptide extension can be inserted between amino acids G20 and C21 of the parent interferon

[0093] In still other embodiments, the parent interferon can be interferon γ and the peptide extension can be inserted between amino acids C23 and Q24 of the parent interferon

[0094] In yet other embodiments, the parent interferon can be interferon λΐ and the peptide extension can be inserted between amino acids A19 and G20 of interferon λΐ. In still yet other embodiments, the parent interferon can be interferon λ2 and the peptide extension can be inserted between amino acids A25 and V26 of interferon λ2. In still yet other embodiments, the parent interferon can be interferon λ3 and the peptide extension can be inserted between amino acids A21 and V22 of interferon A3.

[0095] In some embodiments, the peptide extension can include an amino acid motif, where the amino acid motif can include one or more glycosylation sites. In some embodiments, the amino acid motif can be

where B 1 is an amino acid residue; B² is a Serine (S) or a Threonine (T); and B³ is a sequence of Zl amino acids, where Zl is an integer from 1 to 8 and each amino acid in the sequence B is independently an amino acid residue. [0096] In some embodiments, B¹ can be any amino acid. In other embodiments, B¹ can be any amino acid except for Proline (P). In an embodiment, B¹ can be a Valine (V). In another embodiment, B¹ can be an Isoleucine (I). In still another embodiment, B¹ can be a Glycine (G). In yet another embodiment, B¹ can be an Alanine (A).

[0097] In some embodiments, B² can be a Serine (S). In other embodiments, B² can be a Threonine (T).

[0098] In some embodiments, B³ can be any amino acid. In an embodiment, B³ can be a Valine (V). In another embodiment, B can be an Isoleucine (I). In still another embodiment, B 3 can be a Glycine (G). In yet another embodiment, B 3 can be an Alanine (A).

1 2 3

[0099] Non-limiting examples of the amino acid motif ΝΒΈ Β¾ι include NI[T/S]V, NI[T/S]I, NI[T/S]A, NI[T/S]G, NA[T/S]V, NA[T/S]I, NA[T/S]A, NA[T/S]G, NV[T/S]V, NV[T/S]I, NV[T/S]A, NV[T/S]G, NG[T/S]V, NG[T/S]I, NG[T/S]A, NG[T/S]G, NI[T/S]VNI[T/S]V, NI[T/S]VNA[T/S]G, NI[T/S]VNV[T/S]V, NI[T/S]ANI[T/S]A, NI[T/S]ANI[T/S]G, and NV[T/S]ANG[T/S]I, where [T/S] represents the presence of either a Threonone (T) or a Serine (S) residue. In some embodiments, the amino acid motif NB¹B²[B³]zi can be NITV.

[0100] In some embodiments, the amino acid motif included in the peptide extension can be [B⁴]z₂NB⁵B⁶[B⁷]z3, where B⁴ is a sequence of Z2 amino acids, Z2 is an integer from 1 to 8, and each amino acid in the sequence B⁴ is independently an amino acid residue; B⁵ is an amino acid residue; B⁶ is a Serine (S) or a Threonine (T); B⁷ is a sequence of Z3 amino acids, wherein Z3 is an integer from 1 to 8, wherein each amino acid in the sequence B is independently an amino acid residue.

[0101] In some embodiments, B⁴ can be any amino acid. In an embodiment, B⁴ can be a Valine (V). In another embodiment, B⁴ can be an Isoleucine (I). In still another embodiment, B⁴ can be a Glycine (G). In yet another embodiment, B⁴ can be an Alanine (A).

[0102] In some embodiments, B⁵ can be any amino acid. In an embodiment, B⁵ can be any amino acid except for Proline (P). In an embodiment, B⁵ can be a Valine (V). In another embodiment, B⁵ can be an Isoleucine (I). In still another embodiment, B⁵ can be a Glycine (G). In yet another embodiment, B⁵ can be an Alanine (A). [0103] In some embodiments, B⁶ can be a Serine (S). In other embodiments, B⁶ can be a Threonine (T).

[0104] In some embodiments, B 7 can be any amino acid. In an embodiment, B 7 η

can be a Valine (V). In another embodiment, B can be an Isoleucine (I). In still another embodiment, B 7 can be a Glycine (G). In yet another embodiment, B 7 can be an Alanine (A). In yet another embodiment, B7 can be an Alanine (A). In yet still another embodiment, B7 can be GG. In yet still another embodiment, B7 can be GGG. In yet still another embodiment, B7 can be GR.

[0105] Examples of the amino acid motif [B⁴]z₂NB⁵B⁶[B⁷]z3 include, but are not limited to, INI[T/S]V, INI[T/S]I, VNI[T/S]A, VNI[T/S]G, VNI[T/S]GR, VNITGG, VNI[T/S]GGG, INA[T/S]V, INA[T/S]G, GNA[T/S]I, INA[T/S]A, GNI[T/S]G, GNA[T/S]G, INA[T/S]G, ANA[T/S]G, ANV[T/S]V, VNV[T/S]I, VNV[T/S]A, ANI[T/S]V, VNV[T/S]G, ING[T/S]V, ANG[T/S]I, VNG[T/S]A, VNG[T/S]G, SNI[T/S]G, ASNI[T/S]G, VNI[T/S]VNI[T/S]V, ANI[T/S]VNA[T/S]G, INI[T/S]VNV[T/S]V, GNI[T/S]ANI[T/S]A, GNI[T/S]ANI[T/S]G, VNI[T/S]GVNI[T/S]GR, VNI[T/S]GGVNI[T/S]GR, VNI[T/S]GGGVNI[T/S]GR, and GNV[T/S]ANG[T/S]I, where [T/S] represents the presence of either a Threonone (T) or a Serine (S) residue. In some embodiments, the amino acid motif [B⁴]z₂NB¾^D[B ']z3 can be VNITG. In other embodiments, the amino acid motif [B⁴]_Z2NB⁵B⁶[B⁷]z3 can be VNITGG. In still other embodiments, the amino acid motif [B⁴]_Z2NB⁵B⁶[B⁷]z3 can be VNITGGG. In yet other embodiments, the amino acid motif [B⁴]_Z2NB⁵B⁶[B⁷]z3 can be VNISGR. In yet still other embodiments, the amino acid motif [B⁴]_Z2NB⁵B⁶[B⁷]z3 can be VNITGVNISGR. In some embodiments, the amino acid motif [B⁴]_Z2NB⁵B⁶[B⁷]z3 can be VNITGG VNIS GR. In other embodiments, the amino acid motif [B⁴]_Z2NB⁵B⁶[B⁷]z3 can be VNITGGGVNISGR.

[0106] The amino acid motif, such as

and [B⁴]_Z2NB⁵B⁶[B⁷]_Z3, can be present one or more times in the peptide extension. In some embodiments, the amino acid motif can be present once, twice, three times, or four times in the peptide extension. In still other embodiments, the amino acid motif can be present five times, six times, seven times, eight times, nine times, or ten times in the peptide extension. In yet other embodiments, the amino acid motif can be present eleven times, twelve times, thirteen times, fourteen times, fifteen times, sixteen times, seventeen times, eighteen times, nineteen times, twenty times, twenty-one times, twenty-two times, twenty-three times, twenty-four times, twenty-five times, twenty-six times, twenty-seven times, twenty-eight times, twenty-nine times, thirty times, or more in the peptide extension.

[0107] In some embodiments, the amino acid motif can be present at least once, at least twice, at least three times, or at least four times in the peptide extension. In still other embodiments, the amino acid motif can be present at least five times, at least six times, at least seven times, at least eight times, at least nine times, or at least ten times in the peptide extension. In yet other embodiments, the amino acid motif can be present at least eleven times, at least twelve times, at least thirteen times, at least fourteen times, at least fifteen times, at least sixteen times, at least seventeen times, at least eighteen times, at least nineteen times, at least twenty times, at least twenty-one times, at least twenty- two times, at least twenty-three times, at least twenty-four times, at least twenty-five times, at least twenty-six times, at least twenty-seven times, at least twenty-eight times, at least twenty-nine times, at least thirty times, or more in the peptide extension.

[0108] In some embodiments, the amino acid motif can include one N-linked glycosylation site. In other embodiments, the amino acid motif can include two, three, four, five, or six N-linked glycosylation sites. In some embodiments, the amino acid motif can include one O-linked glycosylation sites. In other embodiments, the amino acid motif can include two, three, four, five, or six O-linked glycosylation sites. In still other embodiments, the amino acid motif can include one, two, three, four, five, or six N-linked gltycosylation sites and/or one, two, three, four, five, or six O-linked glycosylation sites.

[0109] In some embodiments, the amino acid motif can include at least one N- linked glycosylation site. In other embodiments, the amino acid motif can include at least two, at least three, at least four, at least five, or at least six N-linked glycosylation sites. In some embodiments, the amino acid motif can include at least one O-linked glycosylation sites. In other embodiments, the amino acid motif can include at least two, at least three, at least four, at least five, or at least six O-linked glycosylation sites. In still other embodiments, the amino acid motif can include at least one, at least two, at least three, at least four, at least five, or at least six N-linked gltycosylation sites and/or at least one, at least two, at least three, at least four, at least five, or at least six O-linked glycosylation sites.

[0110] Non-limiting examples of peptide extensions include:

VNITG (SEQ ID NO: 1)

VNITGVNITG (SEQ ID NO: 2)

VNITGVNITGVNITG (SEQ ID NO: 3)

VNITGVNITGVNITGVNITG (SEQ ID NO: 4)

VNITGVNITGVNITGVNITGVNITG (SEQ ID NO: 5)

VNITGVNITGVNITGVNITGVNITGVNITG (SEQ ID NO: 6)

VNITGVNITGVNITGVNITGVNITGVNITGVNITG (SEQ ID NO: 7)

VNITGVNITGVNITGVNITGVNITGVNITGVNITG VNITG (SEQ ID NO: 8)

VNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITG (SEQ ID NO: 9) VNITGVNITGVNITG VNITG VNITG VNITGVNITGVNITGVNITGVNITG (SEQ ID NO: 10)

VNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITG (SEQ ID NO: 11)

VNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITG

(SEQ ID NO: 12)

VNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITG VNITGVNITG (SEQ ID NO: 13)

VNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITG VNITGVNITGVNITGVNITG (SEQ ID NO: 14)

VNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITG VNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITG (SEQ ID NO: 15) VNITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNITGGGV NITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNISGR (SEQ ID NO: 16)

VNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITG VNITG VNISGR (SEQ ID NO: 17)

VNITGGG (SEQ ID NO: 18)

VNITGGGVNITGGG (SEQ ID NO: 19) VNITGGGVNITGGGVNITGGG (SEQ ID NO: 20)

VNITGGGVNITGGGVNITGGGVNITGGG (SEQ ID NO: 21)

VNITGGGVNITGGGVNITGGGVNITGGGVNITGGG (SEQ ID NO: 22)

VNITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNITGGG (SEQ ID NO: 23) VNITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNITGGG (SEQ ID NO: 24)

VNITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNITGGG

(SEQ ID NO: 25)

VNITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNITGGGV NITGGG (SEQ ID NO: 26)

VNITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNITGGGV NITGGGVNITGGG (SEQ ID NO: 27)

VNITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNITGGGV NITGGGVNITGGGVNITGGGVNITGGGVNITGGGVNISGR (SEQ ID NO: 28)

VNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITGVNITG VNITGVNISGR (SEQ ID NO: 29)

VNITGGVNITGGVNITGGVNITGGVNITGGVNITGGVNITGGVNITGGVNITGGVNITG G VNITGG VNITGG VNITGG VNIS GR (SEQ ID NO: 30)

[0111] In some embodiments, in addition to the peptide extension that is inserted at a terminal region, the parent interferon can be further modified to include at least one additional glycosylation site in the amino acid sequence of the parent interferon. Each additional glycosylation site that is introduced in the amino acid sequence of the parent interferon can be by at least one amino acid substitution or at least one combination of amino acid substitutions. As such, a hyperglycosylated interferon variant of a parent interferon can be a parent interferon that has been modified to include a peptide extension inserted at a terminal region and at least one additional glycoyslation site introduced to the amino acid sequence of the parent interferon. For example, a hyperglycosylated interferon variant of interferon alfacon-1 can be a modified interferon alfacon-1 that includes a peptide extension at a terminal region and one or more additional glycosylation sites introduced through amino acid substitution(s) such as D31N, D102N, D108N and E138T, in which the positions of the amino acid substitutions are with reference to sequence alignment numbering set forth in Figure 1.

[0112] In some embodiments, the additional glycosylation sites can be introduced to the parent interferon through amino acid substitution(s) located in a region that consists of the amino acid residues after the first 15 amino acids at the amino-terminus of the parent interferon that excludes any signal peptide in the parent interferon and before the last 15 amino acids at the carboxy-terminus of the parent interferon. In other embodiments, the additional glycosylation sites can be introduced to the parent interferon through amino acid substitution(s) located in a region consisting of the first 15 amino acid residues at the amino- terminus of the parent interferon that excludes any signal peptide in the parent interferon. In still other embodiments, the additional glycosylation sites can be introduced to the parent interferon through amino acid substitution(s) located in a region consisting of the last 15 amino acid residues at the carboxy-terminus of the parent interferon. In some embodiments, the parent interferon can be a Type I interferon, where the additional glycosylation sites can be introduced to the parent Type I interferon through amino acid substitution(s) located at a region consisting of the 15th to 150th amino acids, the 20th to 150th amino acids, the 30th to 140th amino acids, the 40th to 120th amino acids, or the 50th to 110th amino acids of the parent Type I interferon, where any signal peptide in the parent Type I interferon is excluded. In another embodiment, the parent interferon can be a Type II interferon, where the additional glycosylation site(s) can be introduced to the parent Type II interferon through amino acid substitution(s) located at a region consisting of the 16th to 150th amino acids, the 40th to 146th amino acids, the 50th to 120th amino acids, or the 60th to 110th amino acids of the parent Type II interferon, where any signal peptide in the parent Type II interferon is excluded. In another embodiment, the parent interferon can be a Type III interferon, where the additional glycosylation site(s) can be introduced to the parent Type III interferon through amino acid substitution(s) located at a region consisting of the 16th to 150th amino acids, the 20th to 148th amino acids, the 25th to 146th amino acids, or the 30th to 140th amino acids in the parent Type III interferon, where any signal peptide in the parent Type III interferon is excluded. [0113] In some embodiments, in addition to the peptide extension that is inserted at a terminal region, the parent interferon has been further modified to include at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten additional glycosylation sites in the amino acid sequence of the parent interferon. Each additional glycosylation site that is introduced to the parent interferon may be a native glycosylation site that is not present in the parent interferon, or a non-native glycosylation site. For example, interferon alfacon-1 that has a peptide extension inserted in a terminal region can be further modified to include a native glycosylation site by introducing the glycosylation site N31-L32-S33 from interferon ocl4 at the homologous amino acid position of interferon alfacon-1. As another example, interferon alfacon-1 that has a peptide extension inserted in a terminal region can be further modified to include a non-native glycosylation site by introducing the glycosylation site N31-L32-S33 from interferon ocl4 at a nonhomologous amino acid position of interferon alfacon-1, such as (D108-E109-S110). As still another example, interferon ocl4 that has a peptide extension inserted in a terminal region can be further modified to include a non-native glycosylation site by introducing its own N31- L32-S33 glycosylation site at a non-homologous amino acid position of interferon ocl4, for example, (D108-E109-S110). In an embodiment, the glycosylation site being introduced into a parent interferon can be from interferon β. In another embodiment, the glycosylation site being introduced into a parent interferon can be from interferon ocl4.

[0114] In some embodiments, the amino acid sequence of the parent interferon can be modified to include a native glycosylation site and a non-native glycosylation site In some embodiments, the amino acid sequence of the parent interferon can be modified to include at least two, three, four, five, six, seven, eight, nine, or ten native glycosylation sites from the parent interferon, and at least two, three, four, five, six, seven, eight, nine, or ten non-native glycosylation sites.

[0115] In some embodiments, the amino acid sequence of the parent interferon can be modified to include an O-linked glycosylation site. In other embodiments, the amino acid sequence of the parent interferon can be modified to include an N-linked glycosylation. In other embodiments, the amino acid sequence of the parent interferon can be modified to include both O-linked and N-linked glycosylation. [0116] In some embodiments, a hyperglycosylated interferon variant of a parent interferon can further include at least one native glycosylation site from the parent interferon that is glycosylated in the hyperglycosylated interferon variant, but is not glycosylated in the parent interferon.

[0117] In some embodiments, the parent interferon can be a Type I interferon, where the parent Type I interferon can be modified to include a peptide extension inserted in a terminal region such as those described herein, and can be further modified to include at least one amino acid substitution or at least one combination of amino acid substitutions selected from X^X31N, X^IN+X^S+X^S, X²32S+X³33S, X³33N, X⁴52N, X⁵54T, X⁶72N, X⁸76N, X⁹78T, X¹⁰101N, X^U102N, X^U102N+X¹²104T, X¹³108N, X¹³108N+X¹⁴110T, X¹⁵138T, X¹⁷145N and X¹⁶144K+X¹⁷145N, where the positions of the amino acid substitutions are with reference to sequence alignment numbering set forth in Figure 1. As used herein, X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, X⁹, X¹⁰, X¹¹, X¹², X¹³, X¹⁴, X¹⁵, X¹⁶, and X¹⁷ represent the amino acid residue at position 31, 32, 33, 52, 54, 72, 74, 76, 78, 101, 102, 104, 108, 110, 138, 144, and 145 of a parent Type I interferon with reference to the sequence alignment numbering set forth in Figure 1 , respectively.

[0118] In some embodiments, X¹ can be any amino acid. In an embodiment, X¹ can be any amino acid except for Asparagine (N). In another embodiment, X¹ can be an Aspartic acid (D). In still another embodiment, X¹ can be a Lysine (K).

[0119] In some embodiments, X² can be any amino acid. In an embodiment, X² can be any amino acid except for Serine (S). In another embodiment, X can be a Leucine (L).

[0120] In some embodiments, X³ can be any amino acid. In an embodiment, X³ can be any amino acid except for Threonine (T). In another embodiment, X can be a Proline

(P). In still another embodiment, X 3 can be a Serine (S). In yet another embodiment, X 3 can be a Leucine (L). In yet still another embodiment, X can be an Isoleucine (I).

[0121] In some embodiments, X⁴ can be any amino acid. In an embodiment, X⁴ can be any amino acid except for Asparagine (N). In another embodiment, X⁴ can be an Arginine (R). In still another embodiment, X⁴ can be a Serine (S). In yet another embodiment, X⁴ can be a Glycine (G). In yet still another embodiment, X⁴ can be a Glutamine (Q).

[0122] In some embodiments, X⁵ can be any amino acid. In an embodiment, X⁵ can be any amino acid except for Threonine (T). In another embodiment, X⁵ can be an Arginine (R). In still another embodiment, X⁵ can be a Serine (S). In yet another embodiment, X⁵ can be a Leucine (L). In yet still another embodiment, X⁵ can be an Isoleucine (I).

[0123] In some embodiments, X⁶ can be any amino acid. In an embodiment, X⁶ can be any amino acid except for Asparagine (N). In another embodiment, X⁶ can be a Glutamic acid (E). In still another embodiment, X⁶ can be a Valine (V). In yet another embodiment, X⁶ can be an Isoleucine (I). In yet still another embodiment, X⁶ can be a Phenylalanine (F). In yet still another embodiment, X⁶ can be a Serine (S). In yet still another embodiment, X⁶ can be a Methionine (M).

[0124] In some embodiments, X⁷ can be any amino acid. In an embodiment, X⁷ can be any amino acid except for Threonine (T). In another embodiment, X can be an η

Aspartic acid (D). In still another embodiment, X can be a Glutamine (Q). In yet another embodiment, X 7 can be a Lysine (K). In yet still another embodiment, X 7 can be a Serine (S).

[0125] In some embodiments, X 8 can be any amino acid. In an embodiment, X 8

Q

can be any amino acid except for Asparagine (N). In another embodiment, X can be a

Q

Histidine (H). In still another embodiment, X can be a Lysine (K). In yet another embodiment, X 8 can be a Glutamine (Q). In yet still another embodiment, X 8 can be a

Q

Thereonine (T). In yet still another embodiment, X can be a Serine (S).

[0126] In some embodiments, X⁹ can be any amino acid. In an embodiment, X⁹ can be any amino acid except for Threonine (T). In another embodiment, X⁹ can be a Phenylalanine (F). In still another embodiment, X⁹ can be a Proline (P). In yet another embodiment, X⁹ can be a Leucine (L). In still yet another embodiment, X⁹ can be a Tyrosine

(Y).

[0127] In some embodiments, X¹⁰ can be any amino acid. In an embodiment, X¹⁰ can be any amino acid except for Asparagine (N). In another embodiment, X¹⁰ can be a Lysine (K). In still another embodiment, X¹⁰ can be a Glutamic acid (E). In yet another embodiment, X can be an Aspartic acid (D). In still yet another embodiment, X can be a Histidine (H).

[0128] In some embodiments, X¹¹ can be any amino acid. In an embodiment, X¹¹ can be any amino acid except for Asparagine (N). In another embodiment, X¹¹ can be an Aspartic acid (D). In still another embodiment, X¹¹ can be a Serine (S). In yet another embodiment, X¹¹ can be a Threonine (T). In yet still another embodiment, X¹¹ can be an Arginine (R). In yet still another embodiment, X¹¹ can be an Isoleucine (I).

[0129] In some embodiments, X¹² can be any amino acid. In an embodiment, X¹² can be any amino acid except for Threonine (T). In another embodiment, X 12 can be a Serine

(S). In still another embodiment, X 12 can be a Lysine (K). In yet another embodiment, X 12 can be a Leucine (L).

[0130] In some embodiments, X¹³ can be any amino acid. In an embodiment, X¹³ can be any amino acid except for Asparagine (N). In another embodiment, X 13 can be an

Aspartic acid (D). In still another embodiment, X 13 can be a Glutamic acid (E). In yet another embodiment, X 13 can be a Lysine (K).

[0131] In some embodiments, X¹⁴ can be any amino acid. In an embodiment, X¹⁴ can be any amino acid except for Threonine (T). In another embodiment, X¹⁴ can be a Serine (S). In still another embodiment, X¹⁴ can be an Aspartic acid (D). In yet another embodiment, X¹⁴ can be an Arginine (R). In yet still another embodiment, X¹⁴ can be an Asparagine (N).

[0132] In some embodiments, X¹⁵ can be any amino acid. In an embodiment, X¹⁵ can be any amino acid except for Threonine (T). In another embodiment, X¹⁵ can be a Glutamic acid (E). In still another embodiment, X¹⁵ can be a Glycine (G). In yet another embodiment, X¹⁵ can be an Isoleucine (I).

[0133] In some embodiments, X¹⁶ can be any amino acid. In an embodiment, X¹⁶ can be any amino acid except for Lysine (K). In another embodiment, X¹⁶ can be an Asparagine (N). In still another embodiment, X¹⁶ can be an Isoleucine (I). In yet another embodiment, X¹⁶ can be a Leucine (L).

[0134] In some embodiments, X¹⁷ can be any amino acid. In an embodiment, X¹⁷ can be any amino acid except for Asparagine (N). In another embodiment, X 17 can be selected from a Valine (V). In still another embodiment, X can be an Alanine (A). In yet another embodiment, X 17 can be a Glutamic acid (E). In yet still another embodiment, X 17 can be a Serine (S). In yet still another embodiment, X 17 can be a Glycine (G).

[0135] Table 2 provided below sets forth the corresponding amino acid substitution in several Type I interferons for an amino acid substitution described herein, where the positions of the amino acid substitutions are with reference to sequence alighment numbering set forth in Figure 1. The convention "X#Z" is used herein to designate amino acid substitutions, where X designates an existing amino acid in a parent Type I interferon, # indicates the amino acid position in the parent Type I interferon, and Z represents the amino acid that replaces X. For example, D31N indicates the replacement of the Aspartic acid (D) at position 31 with an Asparagine (N). In Table 2, "— " means that the corresponding amino acid substitution is unnecessary for the indicated interferon, and "n/a" means that no amino acid residue is present in the specific amino acid position in the indicated interferon, where the positions of the amino acid substitutions are with reference to sequence alighment numbering set forth in Figure 1. A combination of amino acid substitutions is indicated with a "+," for example, D31N+L32S+P33S indicates replacement of the Aspartic acid (D) at position 31 of the Type I interferon with an Asparagine (N), replacement of the Leucine (L) at position 32 of the Type I interferon with a Serine (S), and replacement of the Proline (P) at position 33 of the Type I interferon with a Serine (S).

[0136] For example, as shown in Table 2, for interferon alfacon-1, the corresponding amino acid substitution of X^X31N is D31N. Reading in conjunction with Table 1, the amino acid substitution D31N in interferon alfacon-1 means that the Aspartic acid (D) at position 25 in the sequence of interferon alfacon-1 is substituted by an Asparagine (N). Further, for interferon alfacon-1, the corresponding amino acid substitution of X 32S is L32S, which means that the Leucine (L) at position 26 in the sequence of interferon alfacon-1 is substituted by a Serine (S). Similarly, for interferon alfacon-1, the corresponding amino acid substitution of X 333T is P33T, the corresponding amino acid substitution of X 333N is P33N, the corresponding amino acid substitution of X⁴52N is R52N, the corresponding amino acid substitution of X⁵54T is I54T, the corresponding amino acid substitution of X 72N is E72N, the corresponding amino acid substitution of X'74T is D74T, the corresponding amino acid substitution of X⁹78T is F78T, the corresponding amino acid substitution of X¹⁰101N is K101N, the corresponding amino acid substitution of Xⁿ102N is

D102N, the corresponding amino acid substitution of X 12104T is S104T, the corresponding amino acid substitution of X 13108N is D108N, the corresponding amino acid substitution of X¹⁴110T is SHOT, the corresponding amino acid substitution of X¹⁵138T is E138T, the corresponding amino acid substitution of X¹⁶144K is N144K, and the corresponding amino acid substitution of X 17145N is V145N. Table 2 also shows that the amino acid substitution

Q

X 76N is not necessary for interferon alfacon-1 because interferon alfacon-1 already has an Asparagine (N) at position 76 (which is position 69 in the sequence of interferon alfacon-1, as shown in Table 1).

[0137] Also shown in Table 2, for interferon β, amino acid substitutions X^X31N, X⁴52N, and X¹³108N are not necessary because interferon β already has an Asparagine (N) at positions 31, 52, and 108 (which are positions 25, 46, 101 in the sequence of interferon β, respectively, as shown in Table 1). Similarly, amino acid substitutions X¹⁴110T and X¹⁶144K are also not necessary for interferon β because interferon β already has a Threonine (T) at position 110 and a Lysine (K) at position 144. Further, reading in conjunction with Table 1, L32S which corresponds to amino acid substitution X²32S in interferon β means that the Leucine (L) at position 26 in the sequence of interferon β is substituted by a Serine (S). Similarly, for interferon β, the corresponding amino acid substitution of X³33T is L33T, the corresponding amino acid substitution of X 33N is L33N, the corresponding amino acid substitution of X⁵54T is R54T, the corresponding amino acid substitution of X⁶72T is I72N, η

the corresponding amino acid substitution of X 74T is Q74T, the corresponding amino acid substitution of X⁹78T is F78T, the corresponding amino acid substitution of X¹⁰101N is D101N, the corresponding amino acid substitution of Xⁿ102N is S102N, the corresponding amino acid substitution of X 12104T is S104T, and the corresponding amino acid substitution of X¹⁵138T is E138T. In addition, Table 2 shows that for interferon β, there is no amino acid residue present at position 145, and thus the amino acid substitution X 17145N cannot be made in interferon β. Table 2. Corresponding amino acid substitutions in Type I interferons

--" means that no corresponding amino acid substitution can be made in the indicated interferon. ¾/a" means that the specific amino acid position is absent in the indicated interferon.

Table 2. Corresponding amino acid substitutions in Type I interferons (Continued)

[0138] In some embodiments, in addition to the modification where a peptide extension is inserted at a terminal region of the parent interferon, the parent Type I interferon can be further modified to include at least one amino acid substitution or at least one combination of amino acid substitutions listed in Table 3.

Table 3. Amino acid substitutions or combinations of amino acid substitutions in some embodiments of the hyperglycosylated interferon variants of a parent interferon

[0139] In some embodiments, the parent interferon can be a Type II interferon. In some embodiments, in addition to the peptide extension that is inserted at a terminal region of the parent Type II interferon, the parent Type II interferon can be further modified to include one or more additional glycosylation sites in the amino acid sequence of the parent Type II interferon. Each additional glycosylation site that is introduced in the amino acid sequence of the parent Type II interferon can be by at least one amino acid substitution or at least one combination of amino acid substitutions. In an embodiment, the parent interferon can be interferon γ. In an embodiment, the parent interferon γ has been further modified to include at least the amino acid substitution D25N. In another embodiment, the hyperglycosylated interferon variant of parent interferon γ can further include the amino acid substitutions P26S+Y27T. In still another embodiment, the parent interferon γ has been further modified to include at least the amino acid substitution V28N. In yet another embodiment, the hyperglycosylated interferon variant of parent interferon γ can further include the amino acid substitutions K29S+E30T.

[0140] In some embodiments, the parent interferon can be a Type III interferon. In some embodiments, in addition to the peptide extension that is inserted at a terminal region of the parent Type III interferon, the parent Type III interferon can be further modified to include one or more additional glycosylation sites in the amino acid sequence of the parent Type III interferon. Each additional glycosylation site that is introduced in the amino acid sequence of the parent Type III interferon can be by at least one amino acid substitution or at least one combination of amino acid substitutions. In some embodiments, the parent interferon can be IFN-λΙ, where the parent IFN-λΙ has been further modified to include at least one amino acid substitution selected from P21N and P27N. In an embodiment, the parent IFN-λΙ has been further modified to include the amino acid substitution P21N. In another embodiment, the hyperglycosylated interferon variant of parent IFN-λΙ can further include the amino acid substitutions V22S+P23T. In still another embodiment, the parent IFN-λΙ has been further modified to include the amino acid substitution P27N. In yet another embodiment, the hyperglycosylated interferon variant of parent IFN-λΙ can further include the amino acid substitution T28S. In other embodiments, the parent interferon is IFN-A2, where the parent ΙΡΝ-λ2 has been further modified to include at least one amino acid substitution selected from P27N and G33N. In an embodiment, the parent ΙΡΝ-λ2 has been further modified to include the amino acid substitution P27N. In another embodiment, the hyperglycosylated interferon variant of parent ΙΡΝ-λ2 can further include the amino acid substitutions V28S+A29T. In still another embodiment, the parent ΙΡΝ-λ2 has been further modified to include the amino acid substitution G33N. In yet another embodiment, the hyperglycosylated interferon variant of parent interferon ΙΡΝ-λ2 can further include the amino acid substitutions A34S+L35T. In still other embodiments, the parent interferon is IFN-A3, where the parent ΙΡΝ-λ3 has been further modified to include at least one amino acid substitution selected from P23N and G29N. In an embodiment, the parent ΙΡΝ-λ3 has been further modified to include the amino acid substitution P23N. In another embodiment, the hyperglycosylated interferon variant of parent ΙΡΝ-λ3 can further include the amino acid substitutions V24S+A25T. In still another embodiment, the parent ΙΕΝ-λ3 has been further modified to include the amino acid substitution G29N. In yet another embodiment, the hyperglycosylated interferon variant of parent interferon ΙΕΝ-λ3 can further include the amino acid substitutions A30S+L31T.

[0141] In some embodiments, a hyperglycosylated interferon variant of a parent interferon comprises an amino acid sequence set forth in any one of SEQ ID NOs: 31-36, 38- 39, 41, 43, 45-57, 59-78, and 103-116. In other embodiments, a hyperglycosylated interferon variant of a parent interferon is an interferon consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 31-36, 38-39, 41, 43, 45-57, 59-78, and 103-116.

[0142] In some embodiments, a hyperglycosylated interferon variant of a parent interferon can have at least about 80% amino acid sequence identity, alternatively at least about 81% amino acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity, alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about 88% amino acid sequence identity, alternatively at least about 89% amino acid sequence identity, alternatively at least about 90% amino acid sequence identity, alternatively at least about 91% amino acid sequence identity, alternatively at least about 92% amino acid sequence identity, alternatively at least about 93% amino acid sequence identity, alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternatively at least about 96% amino acid sequence identity, alternatively at least about 97% amino acid sequence identity, alternatively at least about 98% amino acid sequence identity and alternatively at least about 99% amino acid sequence identity to a hyperglycosylated interferon variant sequence as disclosed herein (for example, SEQ ID NOs: SEQ ID NOs: 31- 36, 38-39, 41, 43, 45-57, 59-78, and 103-116), or any fragment of a hyperglycosylated interferon variant interferon sequence as disclosed herein.

[0143] In some embodiments, the glycosylation of hyperglycosylated interferon variants of a parent interferon is altered compared to that of the parent interferon. In some embodiments, the glycosylation pattern of hyperglycosylated interferon variants of a parent interferon is altered compared to that of the parent interferon. In some embodiments, the hyperglycosylated interferon variants of a parent interferon exhibit increased amount of glycosylation compared to the parent interferon.

Functional features of glycosylated interferons

[0144] In some embodiments, a hyperglycosylated interferon variant of a parent interferon can exhibit one or more of the following properties: increased serum half-life; reduced immunogenicity in vivo; increased functional in vivo half-life; increased stability; reduced degradation by gastrointestinal tract conditions; and improved water solubility. In an embodiment, a hyperglycosylated interferon variant of a parent interferon can have an increased serum half-life compared to a naturally occurring interferon or compared to the parent interferon under substantial similar or the same conditions. In another embodiment, a hyperglycosylated interferon variant of a parent interferon can have an increased AUC compared to a naturally occurring interferon or compared to the parent interferon under substantial similar or the same conditions. The term "serum half-life" is used interchangeably herein with the terms "plasma half-life," and "circulating half-life."

[0145] In some embodiments, the hyperglycosylated interferon variants of a parent interferon can have a serum half-life that is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 100%, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, or at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9- fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40- fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80- fold, at least about 90-fold, at least about 100-fold, at least about 200-fold, at least about 300- fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1000-fold, or more, greater than the serum half-life of the parent interferon under substantial similar or the same conditions. In some embodiments, the extent of the increase in serum half-life of a hyperglycosylated interferon variant of a parent interferon is determined by comparing the serum half-life of the hyperglycosylated interferon variant of the parent interferon to the serum half-life of the parent interferon in human blood or human serum in vivo.

[0146] In some embodiments, the hyperglycosylated interferon variants of a parent interferon can have an AUC that is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 100%, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5- fold, at least 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least 9.5-fold, at least 10-fold, or more, greater than the AUC of the parent interferon when administered under substantially similar or the same conditions.

[0147] The serum half-life of a hyperglycosylated interferon variant of a parent interferon can be readily determined using conventional methods. For example, a hyperglycosylated interferon variant of a parent interferon can be detectably labeled, and be administered to a subject (for example, an experimental non-human animal, or a human subject), and, at various time points following administration of the hyperglycosylated interferon variant, a blood sample is drawn and the amount of detectably labeled hyperglycosylated interferon variant in the blood sample can be determined.

[0148] In some embodiments, the hyperglycosylated interferon variants of a parent interferon can be detected in the serum of the subject after at least about 3 days, at least about 5 days, at least about 7 days, at least about 9 days, at least about 11 days, at least about 13 days, at least about 15 days, at least about 17 days, at least about 19 days, at least about 21 days, at least about 23 days, at least about 25 days, at least about 27 days, at least about 29 days, at least about 31 days, at least about 33 days, at least about 35 days, at least about 37 days, at least about 39 days, at least about 41 days, at least about 43 days, at least about 45 days, at least about 47 days, at least about 49 days, at least about 51 days, or longer after administration. Preparation of a hyperglycosylated interferon variant of a parent interferon

[0149] A hyperglycosylated interferon variant of a parent interferon can be prepared using conventional techniques, including chemical synthesis methods, production by standard recombinant techniques, and combinations thereof. For example, the hyperglycosylated interferon variant can be synthesized using an automated solid-phase tert- butyloxycarbonyl and benzyl protection strategy. A hyperglycosylated interferon variant of a parent interferon can also be synthesized by native chemical ligation using standard methods of chemical synthesis. The purity of synthesized polypeptides may be assessed by reverse- phase high performance liquid chromatography (HPLC) and isoelectric focusing. The primary structures of the ligands may be verified by Edman sequencing methods. Examples of techniques and methods for generating hyperglycosylated interferon variants of a parent interferon have been described in U.S. Patent Publication No. 2006-0182716 and U.S. Patent No. 7,597,884, the contents of which are hereby incorporated by reference in their entirety. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like.

[0150] As used herein, the term "host cell" includes an individual cell or cell culture, which can be or has been a recipient of any recombinant vector(s), or synthetic or exogenous polynucleotide. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change. A host cell includes cells transfected or infected in vivo or in vitro with a recombinant vector or a synthetic or exogenous polynucleotide. The term "recombinant host cell" refers to a host cell that includes one or more recombinant vectors. In some embodiments, a host cell can be a prokaryotic cell. In other embodiments, a host cell can be a eukaryotic cell. Non-limiting examples of recombinant vectors include propagation vectors and expression vectors.

[0151] The terms "DNA regulatory sequences," and "regulatory elements," used interchangeably herein, refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and/or regulate expression of a coding sequence and/or production of an encoded polypeptide in a host cell. [0152] The term "transformation" as used herein refers to a permanent or transient genetic change induced in a cell following introduction of exogenous nucleic acid to the cell. Genetic modification can be accomplished either by incorporation of the new DNA into the genome of the host cell, or by transient or stable maintenance of the new DNA as an episomal element. Where the cell is a mammalian cell, a permanent genetic change is generally achieved by introduction of the DNA into the genome of the cell.

[0153] The term "operably linked," as used herein, refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a coding sequence if the promoter effects transcription or expression of the coding sequence.

[0154] The term "construct," as used herein, refers to a recombinant nucleic acid that has been generated for the purpose of the expression of a specific nucleotide sequence(s), or is to be used in the construction of other recombinant nucleotide sequences. An example of a construct is a recombinant DNA.

[0155] Various chemical synthesis methods can be used to obtain hyperglycosylated interferon variants of a parent interferon described herein. For example, a hyperglycosylated interferon variant can be prepared by using an oligonucleotide synthesizer, wherein oligonucleotides are designed based on the amino acid sequence of the desired interferon variant. The codons can be selected such that they are favored in the host cell in which the recombinant interferon will be produced. For example, several small oligonucleotides coding for portions of the hyperglycosylated interferon variant may be synthesized and assembled by PCR, ligation or ligation chain reaction (LCR). The individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly. Once assembled, the nucleotide sequence encoding the hyperglycosylated interferon variant can be inserted into a recombinant propagation vector to produce a sufficient amount of the polynucleotide encoding the amino acid sequence of a hyperglycosylated interferon variant. Alternatively, once assembled, the nucleotide sequence encoding the hyperglycosylated interferon variant can be inserted into a recombinant expression vector for production of the hyperglycosylated interferon variant in a host cell. [0156] In some embodiments, the polynucleotide encoding the amino acid sequence of a hyperglycosylated interferon variant of a parent interferon can be generated such that at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or more, of the codons are codons that are preferred in human sequences. A non-limiting list of example of codons that are preferred in human is shown in Table 4.

Table 4: Codon Usage in Human. Molecular Cloning: A

Laboratory Manual. Sambrook J. and Russell D. W.

Alanine 6.99 GCU (28.0) GCC (41.6)

GCA (20.0) GCG (10.3)

Arginine 5.28 CGU (8.9) CGC (21.4)

CGA (5.4) CGG (10.4)

AGA (9.9) AGG (l l. l)

Asparagine 3.92 AAU (42.3) AAC (57.7)

Aspartic Acid 5.07 GAU (42.8) GAC (57.2)

Cysteine 2.44 UGU (40.6) UGC (59.4)

Glutamic Acid 6.82 GAA (39.2) GAG (60.7)

Glutamine 4.47 CAA (24.8) CAG (75.2)

Glycine 7.10 GGU (15.8) GGC (35.8)

GGA (24.1) GGG (24.3)

Histidine 2.35 CAU (39.6) CAC (60.4)

Isoleucine 4.50 AUU (33.1 ) AUC (54.0)

AUA (12.9) .

Leucine 9.56 UUA (5.5) UUG (11.5)

CUU (ll.l) CUC (20.8)

CUA (6.5) CUG (44.5)

Lysine 5.71 AAA (38.9) AAG (61.1)

Methionine 2.23 AUG (100)

Phenylalanine 3.84 UUU (41.1) UUC (58.2)

Proline 5.67 CCU (27.3) CCC (35.2)

CCA (25.7) CCG (11.6)

Serine 7.25 UCU (18.3) UCC (23.7)

UCA (12.9) UCG (5.9)

AGU (13.2) AGC (25.9)

Threonine 5.68 ACU (22.4) ACC (40.5)

ACA (25.4) ACG (11.8)

Tryptophan 1.38 UGG (IOO)

Tyrosine 3.13 UAU (40.0) UAC (60.0)

Valine 6.35 GUU (16.4) GUC (25.7)

GUA (9.3) GUG (48.7) [0157] The polypeptide-encoding nucleic acid molecules can be propagated by placing a nucleotide sequence that encodes a hyperglycosylated interferon variant of a parent interferon in a recombinant propagation vector. Various viral and non-viral vectors, including plasmids, bacteriophages (for example, lambda, PI, M13, etc.), cosmids, fosmids, Pl-derived artificial chromosomes (PAC), bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), animal viruses, plant virus, or Human Artificial Chromosomes (HAC) may be used as propagation vectors. The choice of vectors will depend on the type of cell in which propagation is desired and the purpose of propagation, and is within the knowledge of one skilled in the art. Propagation vectors that are useful for amplifying and making large amounts of the desired DNA sequence can be used.

[0158] Recombinant expression vectors can be used to producing the hyperglycosylated interferon variants of a parent interferon described herein in a host cell. For example, a hyperglycosylated interferon variant of a parent interferon can be prepared in a recombinant expression vector comprising a nucleotide sequence that encodes the hyperglycosylated interferon variant. The recombinant expression vector comprising a nucleotide sequence that encodes a hyperglycosylated interferon variant can be prepared using conventional methods and be introduced into a host cell, particularly a eukaryotic cell that is capable of glycosylating proteins. A non-limiting example of methods for producing a hyperglycosylated interferon variant of a parent interferon can include culturing a eukaryotic host cell, where the host cell comprises a subject recombinant expression vector, under conditions that favor production of the hyperglycosylated interferon variant; and isolating the hyperglycosylated interferon variant from the culture. In some embodiments, the hyperglycosylated interferon variant can be isolated and purified to greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%, purity.

[0159] If desirable, a recombinant expression vector that is useful for effective production of a hyperglycosylated interferon variant of a parent interferon can be used. Various expression vectors can be used and the choice of appropriate expression vector is based on the knowledge of one skilled in the art. Many such vectors are available commercially. Introduction of expression vectors into a host cell may use any convenient method, such as calcium-precipitated DNA, electroporation, fusion, transfection, infection with viral vectors, biolistics, etc.

[0160] The recombinant expression vectors can include expression cassettes and/or regulatory sequences ("control sequences" or "control regions") that can effect the expression of a desired polynucleotide to which they are operably linked. Various regulatory sequences can be used, including, but not limited to, promoter sequences and enhancer sequences. The expression vectors can also have restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding a desired protein or other protein.

[0161] Expression cassettes disclosed herein can include a transcription initiation region, a promoter region (for example, a promoter that is functional in a eukaryotic cell), a desired polynucleotide, and a transcriptional termination region. After introduction of the DNA, the cells containing the construct may be selected by means of a selectable marker. Expression cassettes may be introduced into a variety of vectors suitable for eukaryotic host cell expression, such as plasmid; HAC; YAC; vectors derived from animal viruses, such as Moloney's murine leukemia virus, SV40, vaccinia virus, baculovirus, retroviruses, and plant viruses; and the like.

[0162] As discussed previously, a hyperglycosylated interferon variant of a parent interferon can be synthesized in an expression host cell. Various expression host cells can be used. An example of a suitable expression host cell is a eukaryotic cell. Examples of a eukaryotic cell include, but are not limited to; a cell from S. cerevisiae; an insect cell in combination with baculovirus vectors; a mammalian cell, such as COS 7 cell, CHO cell, and HEK293 cell; and the like. When a gene is expressed in eukaryotic cells, the protein product may include post-translational modification.

[0163] The hyperglycosylated interferon variant of a parent interferon may be isolated and purified in accordance with conventional methods known to those skilled in the art. For example, a lysate may be prepared of the expression host cells and the lysate may be purified using HPLC, hydrophobic interaction chromatography (HIC), anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, ultrafiltration, gel electrophoresis, affinity chromatography, and/or other purification techniques.

[0164] Any known assay can be used to determine whether a glycosylated interferon, for example, a hyperglycosylated interferon variant of a parent interferon, has the functions of an interferon. Non-limiting examples of the functions of an interferon include specific binding affinity to interferon receptors and activation of interferon-responsive genes. The assays for detecting the function of interferon include, but not limited to, an in vitro cell- based assay to detect activation of interferon-responsive genes (for example, using a reporter gene operably linked to a promoter containing one or more interferon responsive elements) and the like. Such assays also include KIRA assays for interferon receptor activation activity as described in U.S. Patent Publication No. 2006-0182716 and U.S. Patent No. 7,597,884, the contents of which are hereby incorporated by reference in their entirety.

Pharmaceutical Compositions

[0165] Some embodiments disclosed herein relate to compositions, including pharmaceutical compositions, which can include a therapeutically effective amount of one or more hyperglycosylated interferon variant of a parent interferon disclosed herein. In some embodiments, the compositions can include one or more hyperglycosylated interferon variants of a parent interferon described herein and a pharmaceutically acceptable excipient and/or carrier.

[0166] The terms "physiologically acceptable" and "pharmaceutically acceptable" refer to a carrier, diluent or excipient that does not abrogate the biological activity and properties of the hyperglycosylated interferon variants of a parent interferon disclosed herein.

[0167] As used herein, a "carrier" refers to a compound that facilitates the incorporation of a compound, such as a hyperglycosylated interferon variant of a parent interferon, into cells or tissues. For example, without limitation, dimethyl sulfoxide (DMSO) is a commonly utilized carrier that facilitates the uptake of many organic compounds into cells or tissues of a subject.

[0168] As used herein, a "diluent" refers to an ingredient in a pharmaceutical composition that lacks pharmacological activity but may be pharmaceutically necessary or desirable. For example, a diluent may be used to increase the bulk of a potent drug whose mass is too small for manufacture or administration. It may also be a liquid for the dissolution of a drug to be administered by injection, ingestion or inhalation. A common form of diluent in the art is a buffered aqueous solution such as, without limitation, phosphate buffered saline that mimics the composition of human blood.

[0169] As used herein, an "excipient" refers to an inert substance that is added to a pharmaceutical composition to provide, without limitation, bulk, consistency, stability, binding ability, lubrication, disintegrating ability etc., to the composition. For example, a "diluent" is a type of excipient.

[0170] As used herein, the term "therapeutically effective amount" refers to an amount of an active compound, or pharmaceutical agent, that elicits the biological or medicinal response indicated. For example, a therapeutically effective amount of a hyperglycosylated interferon variants of a parent interferon can be the amount need to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. The biological or medicinal response may occur in a tissue, system, animal or human, and includes alleviation of the symptoms of the disease being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art in light of the detailed disclosure provided herein. The therapeutically effective amount of the hyperglycosylated interferon variants of a parent interferon disclosed herein required will depend on the route of administration, the type of animal, including human, being treated, and the physical characteristics of the specific animal under consideration. The therapeutically effective amount can also depend on factors as weight, diet, concurrent medication; and other factors which those skilled in the medical arts will recognize.

[0171] The pharmaceutical compositions disclosed herein may be manufactured in a manner that is itself known, such as by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or tableting processes. For example, the pharmaceutical compositions can be obtained by reacting the hyperglycosylated interferon variants of a parent interferon disclosed herein with inorganic or organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like. Pharmaceutical compositions will generally be tailored to the specific intended route of administration. Additionally, the active ingredients are contained in an amount therapeutically effective to achieve its intended purpose.

[0172] The compounds used in the pharmaceutical combinations disclosed herein may be provided as salts with pharmaceutically compatible counterions. Suitable additional components of a pharmaceutical composition include, but are not limited to, salts, buffers, solubilizers, stabilizers, detergents, protease-inhibiting agents, and the like.

[0173] Suitable routes of administration may, for example, include oral, rectal, topical transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intranasal, intraocular injections or as an aerosol inhalant.

[0174] In some embodiments, one or more hyperglycosylated interferon variants of a parent interferon are formulated into a preparation suitable for oral administration. For oral preparations, the hyperglycosylated interferon variant can be formulated alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives, and/or flavoring agents.

[0175] In other embodiments, one or more hyperglycosylated interferon variants of a parent interferon are formulated into a preparation suitable for injection. For preparations suitable for injection, the hyperglycosylated interferon variant(s) of a parent interferon can be by dissolved, suspended or emulsified in an aqueous solvent (for example, saline, and the like) or a nonaqueous solvent (for example, vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol); and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

[0176] The pharmaceutical compositions described herein can be administered to a human patient per se, or in pharmaceutical compositions where they are mixed with other active ingredients, as in combination therapy, or carriers, diluents, excipients or combinations thereof. Proper formulation is dependent upon the route of administration chosen. Techniques for formulation and administration of the hyperglycosylated interferon variants of a parent interferon described herein are known to those skilled in the art.

[0177] One may also administer the hyperglycosylated interferon variants of a parent interferon disclosed herein in a local rather than systemic manner, for example, via injection of the hyperglycosylated interferon variants of a parent interferon directly into the infected area, often in a depot or sustained release formulation. Furthermore, one may administer the hyperglycosylated interferon variants of a parent interferon in a targeted drug delivery system, for example, in a liposome coated with a tissue-specific antibody. The liposomes can be targeted to and taken up selectively by the organ.

[0178] The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration. Such notice, for example, may be the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the approved product insert. Compositions that include a hyperglycosylated interferon variant of a parent interferon disclosed herein formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

Methods of Use

[0179] Some embodiments disclosed herein relate to a method of treating and/or ameliorating a disease or condition that can include administering to a subject a therapeutically effective amount of one or more hyperglycosylated interferons variants of a parent interferon described herein, or a pharmaceutical composition that includes one or more hyperglycosylated interferons variants of a parent interferon described herein. [0180] As used herein, the terms "treatment," "treating," and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse affect attributable to the disease. Any alleviation of any undesired signs or symptoms of a disease or condition, to any extent can be considered treatment and/or therapy. "Treatment," as used herein, covers any treatment of a disease in a subject, for example, in a human. "Treatment," as used herein, includes, but is not limited to: (a) increasing survival time; (b) decreasing the risk of death due to the disease; (c) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (d) inhibiting the disease, that is, arresting its development (for example, reducing the rate of disease progression); and (e) relieving the disease, that is, causing regression of the disease. Furthermore, treatment may include acts that may worsen the patient's overall feeling of well- being or appearance.

[0181] As used herein, a "subject" refers to an animal that is the object of treatment, observation or experiment. "Animal" includes cold- and warm-blooded vertebrates and invertebrates such as fish, shellfish, reptiles, and in particular, mammals. "Mammal" includes, without limitation, mice; rats; rabbits; guinea pigs; dogs; cats; sheep; goats; cows; horses; primates; such as monkeys, chimpanzees and apes, and, in particular, humans.

[0182] Some embodiments disclosed herein relate to a method of ameliorating and/or treating fibrotic disorders that can include administering to a subject suffering from fibrotic disorders a therapeutically effective amount of one or more hyperglycosylated interferons variants of a parent interferon described herein, or a pharmaceutical composition that includes one or more hyperglycosylated interferons variants of a parent interferon described herein. In some embodiments, the method can further include administering one or more additional anti-fibrotic agents. Additional anti-fibrotic agents include, but are not limited to, SAPK inhibitors (such as pirfenidone or pirfenidone analogs), TNF antagonists (such as etanercept, infliximab, or adalimumab), TGF-β antagonists (such as GLEEVEC), endothelin receptor antagonists (such as TRACLEER), and the like. [0183] Fibrosis is generally characterized by the pathologic or excessive accumulation of collagenous connective tissue. In some embodiments, the fibrotic disorders can be those diseases or conditions affecting the lung such as idiopathic pulmonary fibrosis, pulmonary fibrosis from a known etiology, liver fibrosis or cirrhosis, cardiac fibrosis, and renal fibrosis. Additional fibrotic disorders include, but are not limited to, collagen disease, interstitial lung disease, human fibrotic lung disease (such as obliterative bronchiolitis, idiopathic pulmonary fibrosis, pulmonary fibrosis from a known etiology, tumor stroma in lung disease, systemic sclerosis affecting the lungs, Hermansky-Pudlak syndrome, coal worker's pneumoconiosis, asbestosis, silicosis, chronic pulmonary hypertension, AIDS- associated pulmonary hypertension, sarcoidosis, and the like), fibrotic vascular disease, arterial sclerosis, atherosclerosis, varicose veins, coronary infarcts, cerebral infarcts, myocardial fibrosis, musculoskeletal fibrosis, post-surgical adhesions, human kidney disease (such as nephritic syndrome, Alport's syndrome, HIV-associated nephropathy, polycystic kidney disease, Fabry's disease, diabetic nephropathy, chronic glomerulonephritis, nephritis associated with systemic lupus, and the like), cutis keloid formation, progressive systemic sclerosis (PSS), primary sclerosing cholangitis (PSC), liver fibrosis, liver cirrhosis, renal fibrosis, pulmonary fibrosis, cystic fibrosis, chronic graft versus host disease, scleroderma (local and systemic), Grave's opthalmopathy, diabetic retinopathy, glaucoma, Peyronie's disease, penis fibrosis, urethrostenosis after the test using a cystoscope, inner accretion after surgery, scarring, myelofibrosis, idiopathic retroperitoneal fibrosis, peritoneal fibrosis from a known etiology, drug-induced ergotism, fibrosis incident to benign or malignant cancer, fibrosis incident to microbial infection (such as viral, bacterial, parasitic and fungal infection), Alzheimer's disease, fibrosis incident to inflammatory bowel disease (including stricture formation in Crohn's disease and microscopic colitis), fibrosis induced by chemical or environmental insult (such as cancer chemotherapy, pesticides, radiation, and the like), and the like.

[0184] Some embodiments disclosed herein relate to a method of ameliorating and/or treating cancer that can include administering to a subject suffering from cancer a therapeutically effective amount of one or more hyperglycosylated interferons variants of a parent interferon described herein, or a pharmaceutical composition that includes one or more hyperglycosylated interferons variants of a parent interferon described herein. In some embodiments, the method can further include administering one or more additional anticancer agents. Examples of additional anti-cancer agents include, but are not limited to, chemotherapeutic agents, radiation agents, bone marrow, biological response modifier, agents that act to reduce cellular proliferation, antimetabolite agents, microtubule affecting agents, hormone modulators, antibodies, and anti-angiogenic agents.

[0185] In some embodiments, the cancer can be a carcinoma. In other embodiments, the cancer can be a sarcoma. In still other embodiments, the cancer can be a tumor, such as a solid tumor. In yet other embodiments, the cancer can be leukemia. In yet still other embodiments, the cancer can be lymphoma.

[0186] Examples of carcinomas include, but are not limited to, esophageal carcinoma; hepatocellular carcinoma; basal cell carcinoma, squamous cell carcinoma (various tissues) ; bladder carcinoma, including transitional cell carcinoma; bronchogenic carcinoma; colon carcinoma; colorectal carcinoma; gastric carcinoma; lung carcinoma, including small cell carcinoma and non-small cell carcinoma of the lung; adrenocortical carcinoma; thyroid carcinoma; pancreatic carcinoma; breast carcinoma; ovarian carcinoma; prostate carcinoma; adenocarcinoma; sweat gland carcinoma; sebaceous gland carcinoma; papillary carcinoma; papillary adenocarcinoma; cystadenocarcinoma; medullary carcinoma; renal cell carcinoma; ductal carcinoma in situ or bile duct carcinoma; choriocarcinoma; seminoma; embryonal carcinoma; Wilm's tumor; cervical carcinoma; uterine carcinoma; testicular carcinoma; osteogenic carcinoma; epithelieal carcinoma; nasopharyngeal carcinoma; etc.

[0187] Examples of sarcomas include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.

[0188] Examples of solid tumors include, but are not limited to, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.

[0189] Examples of leukemias include, but are not limited to, chronic myeloproliferative syndromes; acute myelogenous leukemias; chronic lymphocytic leukemias, including B-cell CLL, T-cell CLL prolymphocytic leukemia, and hairy cell leukemia; and acute lymphoblastic leukemias.

[0190] Examples of lymphomas include, but are not limited to, B-cell lymphomas, such as Burkitt's lymphoma; Hodgkin's lymphoma; and the like.

[0191] Some embodiments disclosed herein relate to a method of inhibiting the growth of a tumor that can include administering to a subject having the tumor a therapeutically effective amount of one or more hyperglycosylated interferons variants of a parent interferon described herein, or a pharmaceutical composition that includes one or more hyperglycosylated interferons variants of a parent interferon described herein.

[0192] Some embodiments disclosed herein relate to a method of ameliorating and/or treating viral infection that can include administering to a subject suffering from viral infection a therapeutically effective amount of one or more hyperglycosylated interferons variants of a parent interferon described herein, or a pharmaceutical composition that includes one or more hyperglycosylated interferons variants of a parent interferon described herein. In some embodiments, the method can further include administering one or more additional anti-viral agents. Examples of anti-viral agents include, but are not limited to nucleoside analogs (for example, ribavirin, viramidine and levovirin) and HCV NS3 inhibitors.

[0193] In some embodiments, the viral infection can be caused by a virus selected from an adenovirus, an Alphaviridae, an Arbovirus, an Astrovirus, a Bunyaviridae, a Coronaviridae, a Filoviridae, a Flaviviridae, a Hepadnaviridae, a Herpesviridae, an Alphaherpesvirinae, a Betaherpesvirinae, a Gammaherpesvirinae, a Norwalk Virus, an Astroviridae, a Caliciviridae, an Orthomyxoviridae, a Paramyxoviridae, a Paramyxoviruses, a Rubulavirus, a Morbilli virus, a Papovaviridae, a Parvoviridae, a Picornaviridae, an Aphthoviridae, a Cardioviridae, an Enteroviridae, a Coxsackie virus, a Polio Virus, a Rhinoviridae, a Phycodnaviridae, a Poxviridae, a Reoviridae, a Rotavirus, a Retroviridae, an A-Type Retrovirus, an Immunodeficiency Virus, a Leukemia Viruses, an Avian Sarcoma Viruses, a Rhabdo viruses, a Rubiviridae and/or a Togaviridae. In some embodiments, the viral infection can be a hepatitis C viral infection. In other embodiments, the viral infection can be an HIV infection.

[0194] Some embodiments provides a method of reducing the risk of viral infection for a subject who has been exposed to a virus (for example, a subject who has come into contact with another subject infected with a virus). The method can include administering to the subject who has been exposed to a virus a therapeutically effective amount of one or more hyperglycosylated interferons variants of a parent interferon described herein, or a pharmaceutical composition that includes one or more hyperglycosylated interferons variants of a parent interferon described herein. In some embodiments, the method can further include administering one or more additional anti- viral agents.

[0195] In some embodiments, a hyperglycosylated interferon variant of a parent interferon can be exhibit one or more of the following activities: antiproliferative activity, anti-viral activity, and anti-fibrotic activity. Whether a hyperglycosylated interferon variant of a parent interferon exhibits anti-viral activity can be determined using any known assay, including for example, an in vitro cell-based inhibition of viral replication assay described in Patick et al. Antimicrob. Agents Chemother., 1999, 43:2444-2450. Whether a hyperglycosylated interferon variant of a parent interferon exhibits anti-proliferative activity can be determined using any known assay, including for example, an in vitro cell-based inhibition of proliferation assay.

[0196] As will be readily apparent to one skilled in the art, the useful in vivo dosage to be administered and the particular mode of administration will vary depending upon the age, weight, the severity of the affliction, and animal species treated, the particular hyperglycosylated interferon variant of a parent interferon employed, and the specific use for which these hyperglycosylated interferon variants of a parent interferon are employed. The determination of effective dosage levels, that is the dosage levels necessary to achieve the desired result, can be accomplished by one skilled in the art using routine pharmacological methods. Typically, human clinical applications of products are commenced at lower dosage levels, with dosage level being increased until the desired effect is achieved. Alternatively, acceptable in vitro studies can be used to establish useful doses and routes of administration of the compositions identified by the present methods using established pharmacological methods.

[0197] Although the exact dosage will be determined on a drug-by-drug basis, in most cases, some generalizations regarding the dosage can be made. In some embodiments, the hyperglycosylated interferon variants of a parent interferon disclosed herein may be administered orally or via injection at a dose of between about 0.001 mg and about 30 mg of each ingredient, or between about 0.001 mg and about 25 mg, between about 0.001 mg and about 20 mg, between about 0.001 mg and about 15 mg, between about 0.001 mg and about 10 mg, between about 0.005 mg and about 5 mg, between about 0.005 mg and about 4 mg, between about 0.005 mg and about 3 mg, between about 0.005 mg and about 2 mg, between about 0.005 mg and about 1 mg, preferably between 0.01 mg and 1 mg, for example, between about 0.05 to about 0.5 mg, between about 0.01 to about 0.3 mg, or between about 0.01 to about 0.1 mg.

[0198] In instances where human dosages for the hyperglycosylated interferon variants of a parent interferon have been established for at least some condition, those same dosages, or dosages that are between about 0.1% and 500%, more preferably between about 25% and 250% of the established human dosage can be used. Where no human dosage is established, as will be the case for newly-discovered pharmaceutical compositions, a suitable human dosage can be inferred from ED₅₀ or ID₅₀ values, or other appropriate values derived from in vitro or in vivo studies, as qualified by toxicity studies and efficacy studies in animals.

[0199] In cases of administration of a pharmaceutically acceptable salt, dosages may be calculated as the free base. As will be understood by those of skill in the art, in certain situations it may be necessary to administer the hyperglycosylated interferon variants of a parent interferon disclosed herein in amounts that exceed, or even far exceed, the above- stated, preferred dosage range in order to effectively and aggressively treat particularly aggressive diseases or infections.

[0200] Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the modulating effects, or minimal effective concentration (MEC). The MEC will vary for each hyperglycosylated interferon variant of a parent interferon, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations.

[0201] Dosage intervals can also be determined using MEC value. Compositions should be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.

[0202] It should be noted that the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity or organ dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity). The magnitude of an administrated dose in the management of the disorder of interest will vary with the severity of the condition to be treated and to the route of administration. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and/or dose frequency may also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine.

[0203] In some embodiments, the hyperglycosylated interferon variants of a parent interferon can be administered less frequently at substantially the same amount as compared to the parent interferon to achieve substantially similar or the same therapeutic results. In an embodiment, the hyperglycosylated interferon variants of a parent interferon can be administered orally, once every three months, once every two months, once every month, twice per month, three times per month, once every week, once every five days, once every three days, once every two days, once per day, twice per day, or three times per day substantially continuously or continuously, for the desired duration of treatment. In another embodiment, the hyperglycosylated interferon variants of a parent interferon can be administered via injection, once every three months, once every two months, once every month, twice per month, three times per month, once every week, once every five days, once every three days, once every two days, once per day, twice per day, or three times per day substantially continuously or continuously, for the desired duration of treatment.

[0204] A therapeutic amount of the hyperglycosylated interferon variants of a parent interferon can be administered to a subject at various points of time for a substantial continuous or continuous period of time, for example, for a week or more, or for a month or more, or for a year or more. In some embodiments, the hyperglycosylated interferon variants of a parent interferon is administered for a period of time, which time period can be, for example, at least about 4 weeks, at least about 8 weeks, at least about 12 weeks, at least about 16 weeks, at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks, at least about 54 weeks, at least about 58 weeks, at least about 62 weeks, at least about 64 weeks, at least about 68 weeks, at least about 72 weeks, at least about 74 weeks, at least about 78 weeks, at least about 82 weeks, at least about 86 weeks, at least about 90 weeks, at least about 94 weeks, at least about 98 weeks, at least about 102 weeks, at least about 106 weeks, or longer. For example, the hyperglycosylated interferon variants of a parent interferon can be administered once every two weeks for about six months, once every month for about a year, or twice every month for about two years.

[0205] In other embodiments, the hyperglycosylated interferon variants of a parent interferon can be administered at a dosing interval of about every 3 days to about every 7 days, about every 5 days to every 9 days, about every 7 days to every 11 days, about every 9 days to every 13 days, about every 11 days to every 15 days, about every 13 days to every 17 days, about every 15 days to every 19 days, about every 17 days to every 21 days, about every 19 days to every 23 days, about every 21 days to every 25 days, about every 23 days to every 27 days, about every 25 days to every 29 days, about every 27 days to every 31 days, about every 29 days to every 33 days, about every 31 days to every 35 days, about every 33 days to every 37 days, about every 35 days to every 39 days, about every 37 days to every 41 days, about every 39 days to every 43 days, about every 41 days to every 45 days, about every 43 days to every 47 days, or about every 45 days to every 49 days. [0206] In non-human animal studies, applications of potential products are commenced at higher dosage levels, with dosage being decreased until the desired effect is no longer achieved or adverse side effects disappear. The dosage may range broadly, depending upon the desired effects and the therapeutic indication. Alternatively, dosages may be based and calculated upon the surface area of the subject, as understood by those of skill in the art.

[0207] Hyperglycosylated interferon variants of a parent interferon disclosed herein can be evaluated for efficacy and toxicity using known methods. For example, the toxicology of a particular hyperglycosylated interferon variant of a parent interferon, or of a subset of the hyperglycosylated interferon variants of a parent interferon, sharing certain chemical moieties, may be established by determining in vitro toxicity towards a cell line, such as a mammalian, and preferably human, cell line. The results of such studies are often predictive of toxicity in animals, such as mammals, or more specifically, humans. Alternatively, the toxicity of particular hyperglycosylated interferon variants of a parent interferon in an animal model, such as mice, rats, rabbits, or monkeys, may be determined using known methods. The efficacy of a particular hyperglycosylated interferon variant of a parent interferon may be established using several recognized methods, such as in vitro methods, animal models, or human clinical trials. Recognized in vitro models exist for nearly every class of condition, including but not limited to cancer, cardiovascular disease, and various immune dysfunction. Similarly, acceptable animal models may be used to establish efficacy of chemicals to treat such conditions. When selecting a model to determine efficacy, the skilled artisan can be guided by the state of the art to choose an appropriate model, dose, and route of administration, and regime. Human clinical trials can also be used to determine the efficacy of a hyperglycosylated interferon variant of a parent interferon in humans.

[0208] Some embodiments disclosed herein relate to a method of chemical library screening. In some embodiments, one or more hyperglycosylated interferon variants of a parent interferons can be used in a signal transduction assay to screen for agents that inhibit interferon activities. In an embodiment, a Kinase Receptor Activation (KIRA) Assay as described in International Patent Publication No. WO 1995/14930 is used. The techniques and systems of using KIRA assay for chemical library screening by one or more hyperglycosylated interferon variants of a parent interferon are described in U.S. Patent No. 7,597,884, the contents of which are hereby incorporated by reference in its entirety.

EXAMPLES

[0209] Additional embodiments are disclosed in further detail in the following examples, which are not in any way intended to limit the scope of the claims.

Example 1

Construction of hyperglycosylated variants of interferon alfacon-1 (CIFN)

[0210] This example illustrates the construction of hyperglycosylated variants of interferon alfacon-1 (CIFN) with various amino-terminal peptide extension: (1) a hyperglycosylated variant of the parent CIFN with an N-terminal peptide extension VNITG and additional glycosylation sites at amino acid positions 31, 102, and 108 (herein referred to as "CIFN-Nl-31-102-138"); (2) a hyperglycosylated variant of the parent CIFN with an N- terminal peptide extension (VNITG)₂ and additional glycosylation sites at amino acid positions 31, 102, and 108 (herein referred to as "CIFN-N2-31-102-138"); (3) a hyperglycosylated variant of the parent CIFN with an N-terminal peptide extension (VNITG)₃ and additional glycosylation sites at amino acid positions 31, 102, and 108 (herein referred to as "CIFN-N3-31-102-138"; and (4) a hyperglycosylated variant of the parent CIFN with an N-terminal peptide extension (VNITG)₄ and additional glycosylation sites at amino acid positions 31, 102, and 108 (herein referred to as "CIFN-N4-31-102-138"). Each VNITG motif contains an N-linked glycosylation site.

[0211] N-terminal or C-terminal peptide extensions can be generated through conventional techniques known in the art, such as polymerase chain reaction (PCR)-based mutagenesis. In some experiments, the shorter extension was generated first and the longer extension was generated based on the shorter extension in a step-wise fashion.

[0212] The primers used in the PCR reactions to generate DNA sequences encoding CIFN-Nl-31-102-138, CIFN-N2-31-102-138, CIFN-N3-31-102-138, and CIFN- N4-31-102-138 are listed in Table 5. Table 5. Sequences of primers for generating of hyperglycosylated variants CIFN-N1- 31-102-138, CIFN-N2-31-102-138, CIFN-N3-31-102-138, and CIFN-N4-31-102-138

[0213] To generate DNA sequences encoding CIFN-Nl-31-102-138, a 2-step PCR was used. In step 1 of the PCR reaction, a 506bp fragment 1 was generated by using the gene encoding CIFN-31-102- 138 (which is a variant of the parent CIFN with additional glycosylation sites at amino acid positions 31, 102 and 138) as a template, and CIFN-N1-F and ECOR I-R as primers. A 95bp fragment 2 was generated by using the gene encoding CIFN-31-102-138 as a template, and CIFN-N1-R and HIND III-F as primers. In step 2 of the PCR reaction, fragments 1 and 2 were used as templates to generate a 601bp fragment 3 using ECOR I-R and HIND III-F as primers. Fragment 3 was digested by Hind III and EcoR I, and then cloned into pcDNA3.1 vector (Invitrogen, Carlsbad, CA).

[0214] DNA sequences encoding CIFN-Nl-31-102-138 were generated similarly in which a DNA sequence encoding CIFN-Nl-31-102-138 was used as a template, and CIFN-N2-F and CIFN-N2-R were used as primers in step 1 of the PCR reaction. To synthesize DNA sequences encoding CIFN-N3-31-102-138, a similar 2-step PCR was used in which a DNA sequence encoding CIFN-N2-31-102- 138 was used as a template, and CIFN- N3-F and CIFN-N3-R were used as primers in step 1 of the PCR reaction. To synthesize DNA sequences encoding CIFN-N4-31-102-138, a similar 2-step PCR was used except that DNA sequence encoding CIFN-N3-31-102-138 was used as a template, and CIFN-N4-F and CIFN-N4-R were used as primers in step 1 of the PCR reaction.

[0215] pcDNA3.1 vectors containing DNA sequences encoding each hyperglycosylated variant of CIFN were transfected into mammalian cell lines, such as HEK293 cells, CHO cells and Cos-7 cells, for protein expression.

Example 2

Increased glycosylation and maintained biological activity in hyperglycosylated variants of interferon alfacon-1 (CIFN)

[0216] To analyze the effects of an N-terminal or a C-terminal peptide extension on molecule wight of the parent interferon, DNA encoding various hyperglycosylated variants of CIFN with an N-terminal or a C-terminal peptide extension, such as those from Example 1, were synthesized and transfected into mammalian cell line CHO to express hyperglycosylated variants of CIFN protein. Five days after transfection, the media containing the secreted hyperglycosylated variants of CIFN proteins were collected and analyzed with protein gel electrophoresis and western blot analysis using the polyclonal antibodies targeting the parent CIFN.

1. N-terminal peptide extensions on a CIFN variant (CIFN-31-102-138) increased the molecular weight of the CIFN variant

[0217] Four hyperglycosylated variants of CIFN: CIFN-Nl-31-102-138, CIFN- N2-31-102-138, CIFN-N3-31-102-138, and CIFN-N4-31-102-138, were produced as described in Example 1. CIFN-31-102-138, which is a variant of the parent CIFN having a sequence identical to the four hyperglycosylated variants of the parent CIFN except for the absence of a N-terminal peptide extension, was used as a control in the western blot analysis shown in Figure 4A. As shown in Figure 4A, all 4 hyperglycosylated variants of the parent CIFN had an increased molecular weight compared to the control CIFN-31-102-138. Thus, N-terminal peptide extensions containing various numbers of VNITG motif increased the molecular weight of the control CIFN-31-102-138.

[0218] Also shown in Figure 4A, CIFN-N4-31-102- 138 which had 4 glycosylation sites in the N-terminal peptide extension had the highest molecular weight among the four hyperglycosylated variants of CIFN; CIFN-N3-31-102- 138 which had 3 glycosylation sites in the N-terminal peptide extension had the second highest molecular weight among the four variants; CIFN-N2-31-102-138 which had 2 glycosylation sites in the N-terminal peptide extension had the third highest molecular weight among the four variants; and CIFN-Nl-31-102-138 which had 1 glycosylation site in the N-terminal peptide extension had the lowest molecular weight among the four variants. Therefore, the addition of an N- terminal peptide extension with more glycosylation sites to the CIFN variant CIFN-31-102- 138 increased the molecular weight of CIFN-31-102-138.

2. A C-terminal peptide extension on CIFN variant (CIFN-102-138) increased the molecular weight of the CIFN

[0219] CIFN- 102-138-C2 is a hyperglycosylated variant of CIFN with a C- terminal peptide extension (VNITG)₂ and additional glycosylation sites at amino acid positions 102 and 138. CIFN-102-138 is a variant of CIFN with additional glycosylation sites at amino acid positisions 102 and 138 of the CIFN.

[0220] A DNA sequence encoding CIFN- 102-138-C2 was generated by the PCR- based mutagenesis method as described in Example 1 , and transfected into CHO cells for the expression of CIFN- 102-138-C2 protein. Western blot analysis of Figure 4B showed that CIFN- 102-138-C2 had an increased molecular weight compared to CIFN-102-138. Thus, the C-terminal peptide extension (VNITG)₂ that contains 2 N-linked glycosylated sites increased the molecular weight of CIFN-102-138.

3. Glycosylation of variants of CIFN having a N-terminal peptide extension with 14 glycosylation sites, and l or 2 internal glycosylation sites

[0221] Four hyperglycosylated variants of CIFN having a N-terminal peptide extension with 14 glycosylation sites and 1 or 2 internal glycosylation sites were produced by methods disclosed herein. The four variants are: (1) CIFN-N 14(3)- 102 (SEQ ID NO:31) having the N-terminal peptide extension with 14 glycosylation sites and an additional glysoylation site at amino acid position 102; (2) CIFN-N14(3)-102-138 (SEQ ID NO: 117) having the N-terminal peptide extension with 14 glycosylation sites and two additional glysoylation sites at amino acid positions 102 and 138; (3) CIFN-N14(3)-108-138 (SEQ ID NO: 118) having the N-terminal peptide extension with 14 glycosylation sites and two additional glysoylation sites at amino acid positions 108 and 138; and (4) CIFN-N 14(3)- 108 (SEQ ID NO:l 15) having the N-terminal peptide extension with 14 glycosylation sites and an additional glysoylation site at amino acid position 108.

[0222] As shown in Figure 4C, all 4 hyperglycosylated variants of the parent CIFN had a significantly increased molecular weight compared to the parent CIFN. Thus, the addition of internal glycosylation sites and an N-terminal peptide extension containing additional glycosylation sites to CIFN increased the molecular weight of the starting CIFN. 4. Glycosylation and IFN specific activity of variant of CIFN having additional glycosylation sites at amino acid positions 31, 102 and 138, and various N-terminal peptide extensions

[0223] Four hyperglycosylated variants of CIFN: CIFN-N11-31-102-138, CIFN- N8-31-102-138, CIFN-N6-31-102-138, and CIFN-N4-31-102- 138 were produced similarly by the methods disclosed herein. Other than the additional glycosylation sites at aminio acid positions 31, 102 and 138, CIFN-Nl l-31-102-138 had an N-terminal peptide extension with 11 glycosylation sites, CIFN-N8-31-102-138 had an N-terminal peptide extension with 8 glycosylation sites, CIFN-N6-31-102- 138 had an N-terminal peptide extension with 6 glycosylation sites, and CIFN-N4-31-102- 138 had an N-terminal peptide extension with 4 glycosylation sites. As shown in Figure 4D, all 4 hyperglycosylated variants of the parent CIFN had an increased molecular weight compared to the starting CIFN. Thus, N-terminal peptide extensions containing various numbers of glycosylation sites increased the molecular weight of the starting CIFN.

[0224] Also shown in Figure 4D, CIFN-N11-31-102-138 which had 11 glycosylation sites in the N-terminal peptide extension had the highest molecular weight among the four hyperglycosylated variants of CIFN; CIFN-N8-31-102-138 which had 8 glycosylation sites in the N-terminal peptide extension had the second highest molecular weight among the four variants; CIFN-N6-31-102-138 which had 6 glycosylation sites in the N-terminal peptide extension had the third highest molecular weight among the four variants; and CIFN-N4-31-102-138 which had 4 glycosylation site in the N-terminal peptide extension had the lowest molecular weight among the four variants. Therefore, adding an N-terminal peptide extension with various numbers of glycosylation sites, such as an N-terminal peptide extension with 11, 8, 6 or 4 glycosylation sites, to the CIFN variant CIFN-31-102- 138 increased the molecular weight of CIFN. [0225] To identify the source of the molecular weight increase in the hyperglycosylated variants of CIFN, the proteins of CIFN-N11-31-102-138, CIFN-N8-31- 102-138 and CIFN-N6-31-102-138 were digested with (+) or without (-) glycosidase PNGase F. The digested and undigested proteins were then examined by western blot analysis using the CIFN antibody. As shown in Figure 4E, when the carbohydrates were removed from the proteins by the treatment of glycosidase PNGase F, the molecular weight of all three hyperglycosylated variants of CIFN decreased to a level that is similar to the molecular weight of the parent CIFN. This indicates that the increase in the molecular weight of the hyperglycosylated variants of CIFN were largely caused by the addition of carbohydrates, and thus demonstrates that adding an N-terminal peptide extension with various numbers of glycosylation sites and a number of internal glycosylation sites increased the molecular weight of the hyperglycosylated variants of CIFN compared to the parent CIFN.

[0226] Further, the interferon specific activity of the parent CIFN and hyperglycosylated variants CIFN-N11-31-102-138, CIFN-N8-31-102- 138, CIFN-N6-31-102- 138, and CIFN-N4-31-102-138 were measured in an IFN gene reporter assay using iLite human interferon alpha kit (PBL). As shown in Table 6, all 4 hyperglycosylated variants of CIFN had the interferon specific activity comparable to that of the parent CIFN. This demonstrates that biological potencies of these hyperglycosylated variants of CIFN are similar to that of the parent CIFN.

Table 6. IFN specific activities of CIFN and hyperglycosylated variants of CIFN

3. N-terminal peptide extensions on human interferon beta (IFNB) significantly increased the molecular weight of IFNB

[0227] Various N-terminal peptide extensions were added to human interferon beta (IFNB). IFNB-N10 is a hyperglycosylated variant of IFNB with an N-terminal peptide extension (VNITG)io. IFNB-N8 is a hyperglycosylated variant of IFNB with an N-terminal peptide extension (VNITG)s. IFNB-N6 is a hyperglycosylated variant of IFNB with an N- terminal peptide extension (VNITG)₆. IFNB-N4 is a hyperglycosylated variant of IFNB with an N-terminal peptide extension (VNITG)₄. Each VNITG motif contains an N-linked glycosylation site.

[0228] DNA sequences encoding IFNB-N10, IFNB-N8, IFNB-N6, and IFNB-N4 were generated by the PCR-based mutagenesis method as described in Example 1, and transfected into CHO cells for protein expression, respectively. Western blot analysis of Figure 4F showed that all four hyperglycosylated variants of IFNB with an N-terminal peptide extension had increased molecular weights compared to the parent IFNB. Thus, the additions of N-terminal peptide extensions containing various numbers of VNITG motifs increased the molecular weight of IFNB compared to the parent IFNB.

[0229] Figure 4F shows that IFNB-N10 which had 10 glycosylation sites in the N- terminal peptide extension had the highest molecular weight among the four variants; IFNB- N8 which had 8 glycosylation sites in the N-terminal peptide extension had the second highest molecular weight among the four variants; IFNB-N6 which had 6 glycosylation sites in the N-terminal peptide extension had the third highest molecular weight among the four variants; and IFNB-N4 which had 4 glycosylation sites in the N-terminal peptide extension had the lowest molecular weight among the four variants. Therefore, the addition of an N- terminal peptide extension with more glycosylation sites to the parent IFNB led to a higher increase in the molecular weight of the parent IFNB.

[0230] To identify the source of the molecular weight increase in the hyperglycosylated variants of IFNB, the proteins of IFNB-N10, IFNB-N8 and IFNB-N6 were digested with (+) or without (-) glycosidase PNGase F. The digested and undigested proteins were then examined with western blot analysis using the IFNB antibody. [0231] As shown in Figure 4G, when the carbohydrates were removed from the proteins by the treatment of glycosidase PNGase F, the molecular weight of IFNB-N10, IFNB-N8 and IFNB-N6 decreased to a level similar to the molecular weight of the parent IFNB. These results indicated that the increase in the molecular weight of the hyperglycosylated variants of IFNB were largely contributed by the addition of carbohydrates from the VNITG motif in the N-terminal extension.

[0232] Interferon specific activities of the parent IFNB and hyperglycosylated variants IFNB-N6 and IFNB-N10 were measured by the EMCV/A549 CPE assay. The day before the experiment, the A549 cells were plated at 30,000 cell/well in 96 well plates. On the day of the experiment, test interferons and standard (IFNB) were serially diluted in serum free DMEM media and then added to the plated cells. The wells designated for positive control (cells without virus) and negative control (cells with virus but no drug protection) were not dosed. After 24 hours preincubation with the test interferons or the standard, encephalomyocarditis virus (EMCV) at the MOI of 0.001 was added to the cells, except for the positive controls. The plate was then incubated at 37°C, 5% C0₂ and observed daily for the cytopathic effects (CPE) in the cells. When the negative controls achieved full or near full CPE, the culture media was removed from the wells and Hucker's Crystal Violet Solution was added to the cells and further incubated for 5 minutes at room temperature. The stained plate was them washed to remove the background stain, air dried and scanned. The blue dye were eluted with 33% acetic acid and quantified using ELISA reader. IFNB specific activities of the test interferons were calculated by comparing the 50% CPE of test interferons and the standard. As shown in Tables 7, hyperglycosylated variants IFNB-N6 and IFNB-N10 have an interferon specific activity comparable to that of the parent IFNB.

Table 7. IFNB specific activity of IFNB, IFNB-N6 and IFNB-N10

4. N-terminal peptide extensions on human interferon gamma (IFNG) increased the molecular weight of IFNG

[0233] Two N-terminal peptide extensions having different number of VNITG motif was added to human interferon gamma (IFNG), respectively. IFNG-N5 is a hyperglycosylated variant of IFNG with an N-terminal peptide extension (VNITG)₅. IFNG- N10 is a hyperglycosylated variant of IFNG with an N-terminal peptide extension (VNITG) ₁₀. Each VNITG motif contains an N-linked glycosylation site.

[0234] DNA sequences encoding IFNG-N5 and IFNG-N10 were generated by the PCR-based mutagenesis method as described in Example 1, and transfected into CHO cells for protein expression, respectively. Western blot analysis of Figure 4H showed that both of the hyperglycosylated variants of IFNG with an N-terminal peptide extension had an increased molecular weight compared to the parent IFNG. Thus, additions of N-terminal peptide extensions containing various numbers of VNITG motif increased the molecular weight of IFNG.

[0235] Figure 4H also showed that IFNG-N10 which had 10 glycosylation sites in the N-terminal peptide extension had a higher molecular weight than IFNG-N5 which had 5 glycosylation sites in the N-terminal peptide extension. Therefore, the addition of a peptide extension with more glycosylation sites to the parent IFNG increased the molecular weight of the parent IFNG.

5. An N-terminal peptide extension to IFN alpha 1 (IFNal ), a type I interferon, increased the molecular weight of IFNal

[0236] An N-terminal peptide extension with 10 glycosylation sites was introduced to human interferon alpha 1 (IFNal) using similar techniques described in Example 1 to generate a hyperglycosylated variant IFNal-N10 (SEQ ID NO:l 19). The extent of glycosylation of IFNal and IFNal-N10 was examined by the mobility shift of the proteins in SDS gel electrophoresis.

[0237] As shown in Figure 41, hyperglycosylated variant IFNal -N10 had an increased molecular weight compared to the parent IFNal. Thus, the N-terminal peptide extension containing 10 glycosylation sites increased the molecular weight of the parent IFNal. 6. An N-terminal peptide extension to type III interferons, increased the molecular weight of the type III interferons

[0238] An N-terminal peptide extension with 6 glycosylation sites was introduced to human interferon λΐ, λ2 and λ3 similar techniques described in Example 1 to generate hyperglycosylated variants IFN λ1-Ν6 (SEQ ID NO: 129), IFN λ2-Ν6 (SEQ ID NO: 130) and IFN λ3-Ν6 (SEQ ID NO: 131). The extent of glycosylation of each protein was examined by the mobility shift of the protein in SDS gel electrophoresis.

[0239] As shown in Figure 4J, all hyperglycosylated variants had an increased molecular weight compared to its parent IFN λ. Thus, N-terminal peptide extensions containing glycosylation sites increased the molecular weight of the parent IFN λ.

Example 3

Biological activities of hyperglycosylated variants of CIFN

[0240] Four hyperglycosylated variants of CIFN: CIFN-N14(3)-102, CIFN- N14(3)-108, CIFN-N14(3)-102-138 and CIFN-N14(3)-108-138 were produced similarly by the techniques described in Example 1. These hyperglycosylated variants were assayed for HCV replicon activity and interferon specific activity.

1. HCV replicon activity

[0241] HCV replicon cells were plated in 96 well plates and incubated over night. The interferons were serially diluted and added to the replicon cells the next day. After 3day incubation, the cells were lysed and the luciferase activity was assayed with Bright-glo reagent (Promega). The dose response curves were ploted, and EC50 values were calculated. As shown in Figure 5A-C, the HCV replicon activity of all four hyperglycosylated variants were comparable to that of the parent CIFN. This demonstrates that these hyperglycosylated variants of CIFN are as biologically potent as the parent CIFN.

2. Interferon specific activity

[0242] The day before the experiment, the A549 cells were plated at 30,000 cell/well in 96 well plates. On the day of the experiment, test interferons and standard (CIFN) were serially diluted in serum free DMEM media and then added to the plated cells. The wells designated for positive control (cells without virus) and negative control (cells with virus but no drug protection) were not dosed. After 24 hours preincubation with the test interferons or the standard, encephalomyocarditis virus (EMCV) at the MOI of 0.001 was added to the cells, except for the positive controls. The plate was then incubated at 37°C, 5% C0₂ and observed daily for the cytopathic effects (CPE) in the cells. When the negative controls achieved full or near full CPE, the culture media was removed from the wells and Hucker' s Crystal Violet Solution was added to the cells and further incubated for 5 minutes at room temperature. The stained plate was them washed to remove the background stain, air dried and scanned. The blue dye were eluted with 33% acetic acid and quantified using ELISA reader. Interferon specific activities of the test interferons were calculated by comparing the 50% CPE of test interferons and the standard.

[0243] All four hyperglycosylated variants of CIFN showed comparable interferon specific activity to the parent CIFN (Figure 5D), demonstrating that these hyperglycosylated variants of CIFN are as biologically potent as the parent CIFN.

Example 4

Increased glycosylation in hyperglycosylated variants of interferon beta (IFNB)

[0244] To analyze the effects of an N-terminal peptide extension on molecular weight of a parent interferon, DNA encloding various hyperglycosylated variants of the parent IFNB with an N-terminal peptide extension having 10 glycosylation sites and various signal peptides were synthesized and transfected into mammalian cell line CHO to express hyperglycosylated variants of IFNB protein. Other than the N-terminal peptide extension, EPO-N10 had the signal peptide from EPO, CD33-N10 had the signal peptide from CD33, IFNB-N10 had the signal peptide from IFNB, and DS-N10 had an artificial signal peptide. Several days after transfection, the media containing the secreted hyperglycosylated variants of IFNB proteins were collected. The extent of changes in molecular weight of each protein was determined by the mobility shift of the protein in SDS gel electrophoresis using the polyclonal antibodies targeting the parent IFNB.

[0245] As shown in Figure 6, all hyperglycosylated variants of IFNB with the N- terminal peptide extension with 10 glycosylation sites have increased molecular weight regardless of the signal peptide used. This indicates that hybrid interferons with foreign signal peptide were properly secreted and had significant increased molecular weight. Example 5

Pharmacokinetic (PK) and pharmacodynamic (PD) studies of

a mouse homolog of N14-CIFN-108

[0246] A mouse homolog of N14-CIFN-108 (hereafter "mN14-CIFN-108", SEQ ID NO: 120) was generated by techniques similar to what was described in Example 1. When expressed, mN14-CIFN-108 was glycosylated well with a molecular weight close to lOOkD and was active in EMCV/L929 CPE assay.

[0247] Both mN14-CIFN-108 and unglycosylated mouse IFNal (mIFNal) were administered to mice as a single dose (1000 IU/g) subcutaneously. Animals (N=3) were sacrificed at pre-dose and post-dose for unglycosylated mouse IFNal at 0.5, 1, 3, 5, 12, 24, 48, 96, and 168 hours, and mN14-CIFN-108 at 12, 24, 48, 96, and 168 hours. Mouse plasma and liver were harvested and analyzed for PK and PD markers. Mouse IFNal in plasma was quantified by sandwich ELISA and then converted to international units (IU)/ml based on protein specific activity. Plasma β-2 microglobulin was measured with competitive ELISA and liver OAS1 mRNA with Real-Time PCR normalized against GAPDH.

[0248] As shown in Figure 7 A, the activity of the unglycosylated mIFNal control in the subject droped to a baseline level (about 0.5 IU/ml) within approximately the first 10 hours post initial dosing. Comparatively, the activity of mN14-CIFN-108 were well above 50 IU/ml during the same period of time, and continued to be well above 10 IU/ml during the first 100 hours post initial dosing. This demonstrated that mN14-CIFN-108 stayed in the subject for a markedly longer period of time compared to the mIFNal control.

[0249] Table 8 also supports the results shown in Figure 7A. As shown by the data provided in Table 8, the serum half-life (Ti ₂) of mN14-CIFN-108 is about 40 times higher than that of the unglycosylated mIFNal control. This increase in serum half-life indicates that mN14-CIFN-108 stayed in the subject for a markedly longer period of time compared to the mIFNal control. Moreover, mN14-CIFN-108 shows about 33 fold of improvement in the AUC_t and AUC_mf value (AUC means "area under the curve") as compared to the unglycosylated mIFNal control. The AUC value is a measure of drug exposure in a subject. Thus, an interferon with a higher AUC value requires less frequent dosing to a subject to achieve approximately the same results as compared to an interferon with a lower AUC value. Accordingly, the data provided in Table 8 indicate that a subject receiving mN14-CIFN-108 can be dosed less frequently compared to a subject receiving the same amount of the unglycosylated mIFNal control. For example, a subject could be dosed approximately twice a month with the mN14-CIFN-108 compared to the daily dosing needed with the unglycosylated mIFN al control to achieve similar clinical results.

Table 8. Improved pharmacokinetics profiles of mN14-CIFN-108 compared to the unglycosylated mIFN al control

[0250] In a pharmacodynamic study, beta2-microglobulin protein level and induction of OASl mRNA in liver are two commonly used biomarkers for the efficacy of interferon treatment. The longer the beta2-microglobulin protein level (or the induction of OASl mRNA in liver) stays above the baseline level, the interferon is active for a longer period of time. As shown in Figure 7B, the beta2-microglobulin protein level in the subject administered with the unglycosylated mIFNal droppped to a level that was less than 0.2 fold increase over baseline level within approximately 50 hours post initial dosing. Comparatively, the beta2-microglobulin protein level in the subject administered with mN14- CIFN-108 were well above 1 fold increase over baseline level with the same period of time and were well over 0.5 fold increase until 100 hours after initial dosing. Also shown in Figure 7B, the induction of OASl mRNA in liver dropped significantly to less than 0.1 fold increase over baseline in subjects administered with the unglycosylated mIFN al. In comparison, the induction stayed well above 0.5 fold increase for 100 hours after initial dosing in the subjects administered with mN14-CIFN-108. This elongated-PD response in animals by mN14-CIFN-108 as compared to the unglycosylated mIFNal indicated that mN14-CIFN-108 remained efficacious in the subject for much longer period of time as compared to the unglycosylated mIFNal. These results further supported that a subject receiving mN14-CIFN-108 can be dosed less frequently compared to a subject receiving the same amount of the unglycosylated mIFN al control.

Example 6

Construction of hyperglycosylated variants of interferon alfacon-1 (CIFN)

[0251] This example illustrates the construction of two hyperglycosylated variants of the parent interferon alfacon-1 (CIFN): (1) a hyperglycosylated variant of the parent CIFN with amino acid substitutions D31N and P33S (herein referred to as "CIFN D31N+P33S") and a hyperglycosylated variant of the parent CIFN with amino acid substitutions D31N, L32S and P33T (herein referred to as "CIFN D31N+ L32S+P33T").

[0252] DNA sequences encoding CIFN D31N+P33S and CIFN CIFN D31N+ L32S+P33T were synthesized by site-directed mutagenesis through a two-step polymerase chain reaction (PCR). The primers used in the PCR reactions are listed in Table 9.

Table 9. Sequences of primers for generating of hyperglycosylated variants CIFN D31N+P33S and CIFN D31N+L32S+P33T

[0253] To synthesize DNA sequences encoding CIFN D31N+P33S, in step 1 of the PCR reaction, a 490bp fragment 1 was generated by using the gene encoding CIFN as a template, and CIFN D31N+P33S-F and ECOR I-R as primers. A 90bp fragment 2 was generated by using the gene encoding CIFN as a template, and CIFN D31N+P33S-R and HIND III-F as primers. In step 2 of the PCR reaction, fragments 1 and 2 were used as templates, and ECOR I-R and HIND III-F were used as primers to generate a 584bp fragment 3. Fragment 3 was digested by Hind III and EcoR I, and then cloned into pcDNA3.1 vector (Invitrogen, Carlsbad, CA). DNA sequences encoding CIFN D31N+L32S+P33T were synthesized similarly using the primers CIFN D31N+L32S+P33T-F and CIFN D31N+L32S+P33T-R in the PCR reactions.

[0254] pcDNA3.1 vectors containing DNA sequences encoding CIFN D31N+P33S or CIFN D31N+ L32S+P33T were transfected into mammalian cell lines, such as HEK293 cells, CHO cells and Cos-7 cells, for the expression of CIFN D31N+P33S and CIFN D31N+L32S+P33T proteins.

Example 7

Increased glycosylation in hyperglycosylated variants of interferon alfacon-1 (CIFN)

[0255] Glycosylation of the parent CIFN and a number of hyperglycosylated variants of the parent CIFN was examined using western blot analysis using anti-CIFN polyclonal antibody. The extent of glycosylation of each protein was determined by the mobility shift of the protein in SDS gel electrophoresis.

[0256] Amino acid substitutions D31N and P33S were introduced to CIFN using site-directed mutagenesis technique described in Example 1 to generate the hyperglycosylated variant CIFN D31N+P33S. Similary, amino acid substitutions D31N, L32S and P33T were introduced to CIFN to generate the hyperglycosylated variant CIFN D31N+L32S+P33T; amino acid substitution D108N was introduced to CIFN to generate the hyperglycosylated variant CIFN D108N; and amino acid substitutions D108N and SHOT were introduced to CIFN to generate the hyperglycosylated variant CIFN D108N+S 110T.

[0257] As shown in Figure 8A, the variant CIFN D31N+P33S had little change in gel mobility shift compared to the parent CIFN, suggesting CIFN D31N+P33S was not glycosylated. As shown in Figure 8B, the variant CIFN D31N+L32S+P33T showed a significant change in gel mobility shift compared to the parent CIFN, indicating CIFN D31N+L32S+P33T had increased glycosylation compared to the parent CIFN. As shown in Figure 8B, about 50% of the CIFN D31N+L32S+P33T protein was glycosylated as determined by western blot analysis.

[0258] As shown in Figure 8C, only a small percentage of the CIFN D108N protein was glycosylated. Glycosylation was optimized by introducing an additional amino acid substitution, SHOT. As shown in Figure 8D, the variant CIFN D108N+S110T showed a significant change in gel mobility shift compared to the parent CIFN, indicating CIFN D108N+S110T had increased glycosylation compared to the parent CIFN. As shown in Figure 8D, about 90% of the CIFN D108N+S110T protein was glycosylated as determined by western blot analysis.

[0259] CIFN D31N+L32S+P33T and CIFN D108N+S110T were assayed for hepatitis C virus (HCV) replicon activity using the ELISA-based NPT II assay as described in Tan et al. Hepatology, 2004, 40:407A. The GS4.1 cell line carrying HCV replicons were cultured as described in Guo et al. J. Virol., 2003, 77:10769-10779. GS4.1 cells were plated at about 5000 cells/well in 96-well plates. After 1 day, serially-diluted interferons were added to each well. The cells were then incubated at 37°C with 5% C0₂. After 48 hours, the NPT II ELISA assay was performed to assess the level of HCV replicon in each well. EC₅₀ values were calculated using GraphPadPrism (GraphPad Software, San Diego, CA). CIFN D31N+L32S+P33T and CIFN D108N+S110T both showed virtually identical activity as the parent CIFN in the HCV replicon assay, indicating that these hyperglycosylated variants of CIFN are as biologically potent as the parent CIFN.

Example 8

Pharmacokinetic study of a hyperglycosylated variant of

interferon alfacon-1 (CIFN)

[0260] Amino acid subsitutions P33N, K101N, D108N, SHOT, N144K, and V145N were introduced to the parent CIFN using site-directed mutagenesis technique described in Example 1 to generate the hyperglycosylated variant CIFN P33N+K101N+D108N+S110T+N144K+V145N (herein referred as "CIFN variant-6", SEQ ID NO: 304).

[0261] DNA sequences encoding the CIFN variant-6 were generated by site- directed mutagenesis method described in Example 4. To express the CIFN variant-6 protein, HEK293 cells was transiently transfected with pcDNA3.1 vectors containing DNA sequences encoding the CIFN variant-6. After purification, the CIFN variant-6 protein was size fractionated. As shown in Figure 9A protein gel stained with Coomassie Blue, three fractions of the CIFN variant-6 protein were obtained through protein purification. Fractions 1- 3 corresponded in size to the CIFN variant-6 in which 2, 3, and 4 glycosylation sites are glycosylated, respectively. Western blots confirmed the glycosylation by PNGase F digestion (Figures 9B-C). The fractions were quantified by the Bradford method, and named as 4-Gly CIFN variant-6, 3-Gly CIFN variant-6, and 2-Gly CIFN variant-6, respectively.

[0262] In the rat pharmacokinetic study, each of the 4 cohorts had 3 rats. Each of the 4 cohorts were subcutaneously dosed with 4-Gly CIFN variant-6, 3-Gly CIFN variant-6,

2- Gly CIFN variant-6, or the parent CIFN control. The rats' plasma were collected at various time points over a period of 96 hours and examined for blood IFN activity by a gene-reporter assay. Figure 10 shows the blood IFN activity over time. The Tmax; Cmax, Τχ/₂, and AUCinf were determined and are provided in Table 10.

[0263] As shown in Figure 10, the activity of the parent CIFN control in the subject droped to a baseline level (< 5 IU/ML) within approximately the first 10 hours post initial dosing. Comparatively, the activities of 4-Gly CIFN variant-6, 3-Gly CIFN variant-6 and 2-Gly CIFN variant-6 were well above 100 IU/ML during the same time period. As shown in Figure 10, the activity of the 4-Gly CIFN variant-6 continued to be markedly higher than the same baseline level of the parent CIFN for at least 80 hours post initial dosing. Similarly, the activity of the 3-Gly CIFN variant-6 was well above 10 IU/ML at approximately 40 hours post initial dosing. As shown in Figure 10, the activity of the 3-Gly CIFN variant-6 continued to be markedly higher than the same baseline level of the parent CIFN for more than 60 hours after initial dosing. The 2-Gly CIFN variant-6 also had similar high and long-lasting activity in the subject. The activity of the 2-Gly CIFN variant-6 in the subject continued to be markedly higher than the same baseline level of the parent CIFN for more than 20 hours post initial dosing. As shown in Figure 10, the activity of the 2-Gly CIFN variant-6 did not reach the same baseline level of the parent CIFN for more than 40 hours post dosing. Taken together, Figure 10 shows that 4-Gly CIFN variant-6, 3-Gly CIFN variant-6 and 2-Gly CIFN variant-6 stayed in the subject for a markedly longer period of time compared to the parent CIFN control.

[0264] Table 10 also supports the results shown in Figure 10. As shown by the data provided in Table 10, the serum half-life (Ti_/2) of 4-Gly CIFN variant-6 is about 11 times higher than that of the parent CIFN, the T ₂ of 3-Gly CIFN variant-6 is more than about 8 times of that of the parent CIFN, and the T ₂ of 2-Gly CIFN variant-6 is nearly 3 times of that of the parent CIFN. This increase in serum half-life indicates that 4-Gly CIFN variant-6, 3-Gly CIFN variant-6, and 2-Gly CIFN variant-6 stayed in the subject for a markedly longer period of time compared to the parent CIFN. Moreover, the 4-Gly CIFN variant-6 shows about a 6-fold improvement in AUCi_nf value (AUC means "area under the curve") as compared to the parent CIFN. Similarly, the 3-Gly CIFN variant-6 and 2-Gly CIFN variant-6 show approximately a 4-fold and 3-fold improvement in AUQ_nf value compared to CIFN, respectively. The AUC value is a measure of drug exposure in a subject. Thus, an interferon with a higher AUC value requires less frequent dosing to a subject to achieve approximately the same results as compared to an interferon with a lower AUC value. Accordingly, the data provided in Table 7 indicate that a subject receiving 4-Gly CIFN variant-6, 3-Gly CIFN variant-6 and 2-Gly CIFN variant-6 could be dosed less frequently compared to a subject receiving the same amount of the parent CIFN. For example, a subject could be dosed approximately once a week with the 4-Gly CIFN variant-6 compared to the daily dosing needed with the parent CIFN to achieve similar clinical results.

Table 10. Improved pharmacokinestic profiles of various fractions of CIFN variant-6 compared to the parent CIFN

Example 9

Yellow fever virus (YFV) study of a hyperglycosylated variant of

interferon alfacon-1 (CIFN)

[0265] To determine the effectiveness of once weekly (QW) dosing of CIFN P33N+K101N+D108N+S110T+N144K+V145N (CIFN variant-6) in treating yellow fever virus (YFV), a hamster model infected with YFV was used. Each of the 5 cohorts had 10 hamsters. Each hamster was injected intraperitoneally (i.p.) with YFV (10 CCID50/animal). The primary end point of the study was the survival rate among 10 animals in each cohort, and the secondary end points of the study were the ATL levels in blood, the viral load reductions in the liver and the serum, and the body weight changes.

[0266] For Cohort 1, the 4-Gly CIFN variant-6 was administered to the animals once every week (QW) for 2 weeks. For Cohort 2, the 3-Gly CIFN variant-6 was administered to the animals twice every week (BIW) for 2 weeks. For Cohort 3, the parent CIFN control was administered to the animals once every day (QD) for 2 weeks. For Cohort 4, the Peg-IFN-0C-2a control was administered to the animals once every week (QW) for 2 weeks. For Cohort 5, the phosphate-buffered saline (PBS) control was administered to the animals once every day (QD) for 2 weeks. All compounds were administered intraperitoneally at a dose of 1.25xl0⁶ IU/kg/dose with a concentration of 2.5xl0⁵ IU/ml in lxPBS. As a primary end point, mortality was observed daily for 21 days. As a secondary end point, serum ALT leves were measured 6 days post- virus injection (dpi).

[0267] As a satellite study to measure the viral load reduction, 4 cohorts (5 animals per cohort) were dosed with daily CIFN (daily dosing for 3 days), PBS (daily dosing for 3 days), 3-Gly (single dose) and Peg-IFN-0C-2a (single dose), respectively. All of the 5 animals in each cohort in the satellite study were sacrificed on day 4. The reduction of viral titers in the hamsters' serum and liver was measured 4 days post-virus injection (dpi) for every treatment group except for the cohort receiving the 4-Gly CIFN variant-6.

[0268] As shown in Figure 11, QW administration of the 4-Gly CIFN variant-6 was efficacious in YFV model (p<0.01). The 4-Gly CIFN variant-6 group showed an approximately 90% survival rate as compared to the approximately 20% survivial rate for the control PBS group. Also shown in Figure 11, QW administration of the 4-Gly CIFN variant- 6 and QD administration of the parent CIFN had similar survivial rate. This indicates that a subject could be dosed once a week with the 4-Gly CIFN variant-6 compared to the daily dosing with the parent CIFN needed to achieve a similar therapeutic result. As shown in Figure 12A, while the 3-Gly CIFN variant-6 induced an intermediate ALT response, the ALT levels were normal in hamsters that were administered with the 4-Gly CIFN variant-6 once a week. QW administration of the 4-Gly CIFN variant-6 and Peg-IFN-a-2a, QD administration of the parent CIFN all resulted in a statistically significant lower ALT level compared to the QD administration of the PBS control. This indicates that QW administration of the 4-Gly CIFN variant-6 is as effective as QW administration of Peg-IFN-a-2a and QD administration of the parent CIFN.

[0269] Viral load was measured by a CPE assay 4 days post-virus injection (dpi). As shown in Figures 12B-C, viral titers were siginificantly reduced in both the liver and the serum to levels below detection limit for ΒΓ\Υ administration with the 3-Gly CIFN variant-6, QW administration of Peg-IFN-a-2a and QD administration of the parent CIFN. The data show that ΒΓ\ν administration of the 3-Gly CIFN variant-6 is as effective as QW administration of Peg-IFN-a-2a and QD administration of the parent CIFN.

[0270] It will be understood by those of skill in the art that numerous and various modifications can be made without departing from the spirit of the present disclosure. Therefore, it should be clearly understood that the forms disclosed herein are illustrative only and are not intended to limit the scope of the present disclosure.

Claims

WHAT IS CLAIMED IS:

1. A hyperglycosylated interferon variant of a parent interferon, wherein the parent interferon is selected from the group consisting of a Type I interferon, a Type II interferon, and a Type III interferon, wherein the hyperglycosylated interferon variant is the parent interferon that has been modified to include a peptide extension inserted at a terminal region, wherein the peptide extension is a peptide of 1-200 consecutive amino acids and comprises at least two glycosylation sites, wherein the terminal region is selected from the group consisting of an amino-terminal region that consists of the first 15 amino acids at the amino-terminus of the parent interferon that excludes any signal peptide in the parent interferon and a carboxy-terminal region that consists of the last 15 amino acids at the carboxy-terminus of the parent interferon.

2. The hyperglycosylated interferon variant of claim 1, wherein the peptide extension comprises at least three glycosylation sites.

3. The hyperglycosylated interferon variant of claim 1, wherein the peptide extension comprises at least four glycosylation sites.

4. The hyperglycosylated interferon variant of claim 1, wherein the peptide extension comprises at least six glycosylation sites.

5. The hyperglycosylated interferon variant of claim 1, wherein the peptide extension comprises at least eight glycosylation sites.

6. The hyperglycosylated interferon variant of claim 1, wherein the peptide extension comprises at least ten glycosylation sites.

7. The hyperglycosylated interferon variant of any one of claims 1 to 6, wherein the peptide extension is inserted at the amino-terminal region that consists of the first 15 amino acids at the amino-terminus of the parent interferon that excludes any signal peptide in the parent interferon.

8. The hyperglycosylated interferon variant of any one of claims 1 to 6, wherein the peptide extension is inserted at the carboxy-terminal region that consists of the last 15 amino acids at the carboxy-terminus of the parent interferon.

9. The hyperglycosylated interferon variant of any one of claims 1 to 8, wherein the peptide extension comprises an amino acid motif NB 1 B2 [B 3

]zi, wherein B is an amino acid residue;

B² is Serine (S) or Threonine (T);

B is a sequence of Zl amino acids, wherein Zl is an integer from 1 to 8, wherein each amino acid in the sequence B is independently an amino acid residue.

10. The hyperglycosylated interferon variant of any one of claims 1 to 8, wherein the peptide extension comprises an amino acid motif [B⁴]z₂NB⁵B⁶[B⁷]z₃, wherein

B⁴ is a sequence of Z2 amino acids, wherein Z2 is an integer from 1 to 8, wherein each amino acid in the sequence B⁴ is independently an amino acid residue;

B⁵ is an amino acid residue;

B⁶ is Serine (S) or Threonine (T);

B is a sequence of Z3 amino acids, wherein Z3 is an integer from 1 to 8, wherein each amino acid in the sequence B is independently an amino acid residue.

11. The hyperglycosylated interferon variant of any one of claims 9 to 10, wherein the amino acid motif is present one time in the peptide extension.

12. The hyperglycosylated interferon variant of any one of claims 9 to 10, wherein the amino acid motif is present at least two times in the peptide extension.

13. The hyperglycosylated interferon variant of any one of claims 9 to 10, wherein the amino acid motif is present at least three times in the peptide extension.

14. The hyperglycosylated interferon variant of any one of claims 9 to 10, wherein the amino acid motif is present at least four times in the peptide extension.

15. The hyperglycosylated interferon variant of any one of claims 9 to 10, wherein the amino acid motif is present at least six times in the peptide extension.

16. The hyperglycosylated interferon variant of any one of claims 9 to 10, wherein the amino acid motif is present at least eight times in the peptide extension.

17. The hyperglycosylated interferon variant of any one of claims 9 to 10, wherein the amino acid motif is present at least ten times in the peptide extension.

18. The hyperglycosylated interferon variant of any one claims 10 to 17, wherein B⁴ is Valine (V), Isoleucine (I), Glycine (G), or Alanine (A);

B⁵ is Valine (V), Isoleucine (I), Glycine (G), or Alanine (A); and η

B is is a sequence selected from the group consisting of G, GG, GGG, and

GR.

19. The hyperglycosylated interferon variant of claim 18, wherein

B⁴ is Valine (V) or Isoleucine (I); and

B is a sequence selected from the group consisting of G, GG, GGG, and GR.

20. The hyperglycosylated interferon variant of claim 19, wherein the amino acid motif is VNITG.

21. The hyperglycosylated interferon variant of claim 19, wherein the amino acid motif is VNITGG.

22. The hyperglycosylated interferon variant of claim 19, wherein the amino acid motif is VNITGGG.

23. The hyperglycosylated interferon variant of claim 19, wherein the amino acid motif is VNISGR.

24. The hyperglycosylated interferon variant of any one of claims 1 to 23, wherein the parent interferon is a Type I interferon.

25. The hyperglycosylated interferon variant of claim 24, wherein the Type I interferon is selected from the group consisting of interferon a (IFN a), interferon β (IFN β), interferon κ (IFN κ), interferon ω (IFN ω), and interferon τ (IFN X).

26. The hyperglycosylated interferon variant of claim 25, wherein the parent interferon has been further modified to include at least one additional glycosylation site, wherein at least one additional glycosylation site is introduced by an amino acid substitution selected from the group consisting of X^X31N, X³33N, X⁴52N, X⁵54T, X⁶72N, X⁸76N, X⁹78T, X¹⁰101N, X^U102N, X¹³108N, X¹⁵138T, and X¹⁷145N, wherein

X¹ is selected from the group consisting of an Aspartic acid (D) and a Lysine (K);

X is selected from the group consisting of a Proline (P), a Serine (S), a Leucine (L), and a Isoleucine (I);

X⁴ is selected from the group consisting of a Serine (S), an Arginine (R), a Glycine (G), and a Glutamine (Q);

X⁵ is selected from the group consisting of an Arginine (R), an Isoleucine (I), a Serine (S), and a Leucine (L); X⁶ is selected from the group consisting of a Glutamic acid (E), a Valine (V), a Phenylalanine (F), a Methionine (M), a Serine (S), and an Isoleucine (I);

X is selected from the group consisting of a Histidine (H), a Lysine (K), a Glutamine (Q), a Serine (S) and a Threonine (T);

X⁹ is selected from the group consisting of a Phenylalanine (F), a Proline (P), a Leucine (L), and a Tyrosine (Y);

X¹⁰ is selected from the group consisting of a Lysine (K), a Glutamic acid (E), an Aspartic acid (D), a Histidine (H), and a Glutamic acid (E);

X¹¹ is selected from the group consisting of an Aspartic acid (D), a Serine (S), a Threonine (T), an Arginine (R), and an Isoleucine (I);

X 13 is selected from the group consisting of an Aspartic acid (D), a Glutamic acid (E), and a Lysine (K);

X¹⁵ is selected from the group consisting of a Glutamic acid (E), a Glycine (G), and an Isoleucine (I);

X 17 is selected from the group consisting of an Alanine (A) and a Glutamic acid (E), a Valine (V), a Serine (S), and a Glycine (G);

in which the positions of the amino acid substitutions are with reference to sequence alignment numbering set forth in Figure 1.

27. The hyperglycosylated interferon variant of claim 25, wherein the parent interferon is an interferon a (IFN a).

28. The hyperglycosylated interferon variant of claim 27, wherein the parent interferon has been further modified to include at least one additional glycosylation site, wherein at least one additional glycosylation site is introduced by an amino acid substitution selected from the group consisting of X^X31N, X³33N, X⁴52N, X⁵54T, X⁶72N, X⁸76N, X⁹78T, X¹⁰101N, X^U102N, X¹³108N, X¹⁵138T, and X¹⁷145N,

X^I is an Aspartic acid (D);

X is selected from the group consisting of a Proline (P) and a Serine (S);

X⁴ is selected from the group consisting of a Serine (S), an Arginine (R), and a

Glycine (G);

X⁵ is an Isoleucine (I); X⁶ is selected from the group consisting of a Glutamic (E) and a Valine (V);

X is selected from the group consisting of a Histidine (H) and a Lysine (K);

X⁹ is a Phenylalanine (F);

X¹⁰ is selected from the group consisting of a Lysine (K) and a Glutamic acid (E); X¹¹ is an Aspartic acid (D);

X 13 is selected from the group consisting of an Aspartic acid (D) and a Glutamic acid (E);

X¹⁵ is selected from the group consisting of a Glutamic acid (E), a Glycine (G) and an Isoleucine (I);

X 17 is selected from the group consisting of an Alanine (A), a Glutamic acid (E) and a Valine (V);

29. The hyperglycosylated interferon variant of any one of claims 27 to 28, wherein the interferon a (IFN a) is selected from the group consisting of interferon alfacon-1, interferon a2a, interferon a2b, interferon al, interferon a4a, interferon a4b, interferon a5, interferon a l, interferon a8, interferon alO, interferon al3, interferon al4, interferon al6, interferon al7, interferon a21, interferon aH, interferon al, interferon aJl, recombinant interferon alpha-2b, recombinant interferon alpha-2a, recombinant interferon alpha-2C, interferon alpha-nl, interferon alpha-n3, IFN-con2, and IFN-con3.

30. The hyperglycosylated interferon variant of claim 29, wherein the peptide extension is located between amino acids G29 and C30 of the interferon a, wherein the positions of the amino acid substitutions are with reference to sequence alignment numbering set forth in Figure 1.

31. The hyperglycosylated interferon variant of any one of claims 29 to 30, wherein the parent interferon is interferon alfacon-1.

32. The hyperglycosylated interferon variant of claim 31, wherein interferon alfacon-1 has been modified to include at least one additional glycosylation site, wherein at least one additional glycosylation site is introduced by an amino acid substitution selected from the group consisting of X^X31N, X³33N, X⁴52N, X⁵54T, X⁶72N, X⁹78T, X¹⁰101N, X^U102N, X¹³108N, X¹⁵138T, and X¹⁷145N, wherein X¹ is an Aspartic acid (D), X³ is a Proline (P), X⁴ is an Arginine (R), X⁵ is an Isoleucine (I), X⁶ is a Glutamic acid (E), X⁹ is a Phenylalanine (F), X¹⁰ is a Lysine (K), X¹¹ is an Aspartic acid (D), X¹³ is an Aspartic acid (D), X¹⁵ is a Glutamic acid (E), and X¹⁷ is a Valine (V).

33. The hyperglycosylated interferon variant of claim 32, wherein at least one additional glycosylation site is introduced to interferon alfacon-1 by the amino acid substitution X^X31N, wherein X¹ is an Aspartic acid (D).

34. The hyperglycosylated interferon variant of claim 33, further comprising the amino acid substitutions X 233S and X 333T, wherein X 2 is a Leucine (L) and X 3 is a Serine (S).

35. The hyperglycosylated interferon variant of claim 32, wherein at least one additional glycosylation site is introduced to interferon alfacon-1 by the amino acid substitution X 33 IN, wherein X 3 is a Proline (P).

36. The hyperglycosylated interferon variant of claim 32, wherein at least one additional glycosylation site is introduced to interferon alfacon-1 by the amino acid substitution X⁶72N, wherein X⁶ is a Glutamic acid (E).

37. The hyperglycosylated interferon variant of claim 36, further comprising the amino acid substitution X 774T, wherein X 7 is an Aspartic acid (D).

38. The hyperglycosylated interferon variant of claim 32, wherein at least one additional glycosylation site is introduced to interferon alfacon-1 by the amino acid substitution X⁹78T, wherein X⁹ is a Phenylalanine (F).

39. The hyperglycosylated interferon variant of claim 32, wherein at least one additional glycosylation site is introduced to interferon alfacon-1 by the amino acid substitution X¹⁰101N, wherein X¹⁰ is a Lysine (K).

40. The hyperglycosylated interferon variant of claim 32, wherein at least one additional glycosylation site is introduced to interferon alfacon-1 by the amino acid substitution Xⁿ102N, wherein X¹¹ is an Aspartic acid (D).

41. The hyperglycosylated interferon variant of claim 40, further comprising the amino acid substitution X 12104T, wherein X 12 is a Serine (S).

42. The hyperglycosylated interferon variant of claim 32, wherein at least one additional glycosylation site is introduced to interferon alfacon-1 by the amino acid substitution X 13108N, wherein X 13 is an Aspartic acid (D).

43. The hyperglycosylated interferon variant of claim 42, further comprising the amino acid substitution X¹⁴110T, wherein X¹⁴ is a Serine (S).

44. The hyperglycosylated interferon variant of claim 32, wherein at least one additional glycosylation site is introduced to interferon alfacon-1 by the amino acid substitution X¹⁵138T, wherein X¹⁵ is a Glutamic Acid (E).

45. The hyperglycosylated interferon variant of claim 32, wherein at least one additional glycosylation site is introduced to interferon alfacon-1 by the amino acid substitution X¹⁷145N, wherein X¹⁷ is a Valine (V).

46. The hyperglycosylated interferon variant of claim 45, further comprising the amino acid substitution X¹⁶144K, wherein X¹⁶ is an Asparagine (N).

47. The hyperglycosylated interferon variant of claim 32, wherein at least three additional glycosylation sites are introduced to interferon alfacon-1 by the amino acid substitutions X'31N, X¹¹102N and X°138T, wherein X¹ is an Aspartic acid (D), X is an Aspartic acid (D), and X¹⁵ is a Glutamic Acid (E).

48. The hyperglycosylated interferon variant of claim 32, wherein at least three additional glycosylation sites are introduced to interferon alfacon-1 by the amino acid substitutions X⁹78T, Xⁿ102N and X¹⁵138T, wherein X⁹ is a Phenylalanine (F), X¹¹ is an Aspartic acid (D), and X¹⁵ is a Glutamic Acid (E).

49. The hyperglycosylated interferon variant of any one of claims 29 to 30, wherein the parent interferon is interferon oc2a.

50. The hyperglycosylated interferon variant of claim 49, wherein interferon oc2a has been modified to include at least one additional glycosylation site, wherein at least one additional glycosylation site is introduced by an amino acid substitution selected from the group consisting of X^X31N, X³33N, X⁴52N, X⁵54T, X⁶72N, X⁹78T, X¹⁰101N, Xⁿ102N, X¹³108N, X¹⁵138T, and X¹⁷145N, wherein X¹ is an Aspartic acid (D), X³ is a Proline (P), X⁴ is an Arginine (R), X⁵ is an Isoleucine (I), X⁶ is a Glutamic Acid (E), X⁹ is a Phenylalanine (F) , X¹⁰ is a Lysine (K), X¹¹ is an Aspartic acid (D), X¹³ is an Aspartic acid (D), and X¹⁷ is a Glutamic Acid (E).

51. The hyperglycosylated interferon variant of any one of claims 29 to 30, wherein the parent interferon is interferon oc2b.

52. The hyperglycosylated interferon variant of claim 51, wherein interferon oc2b has been modified to include at least one additional glycosylation site, wherein at least one additional glycosylation site is introduced by an amino acid substitution selected from the group consisting of X^X31N, X³33N, X⁴52N, X⁵54T, X⁶72N, X⁹78T, X¹⁰101N, Xⁿ102N, X¹³108N, and X¹⁷145N, wherein X¹ is an Aspartic acid (D), X³ is a Proline (P), X⁴ is an Arginine (R), X⁵ is an Isoleucine (I), X⁶ is a Glutamic Acid (E), X⁹ is a Phenylalanine (F), X¹⁰ is a Lysine (K), X¹¹ is an Aspartic acid (D), X¹³ is an Aspartic acid (D), and X¹⁷ is a Glutamic Acid (E).

53. The hyperglycosylated interferon variant of any one of claims 29 to 30, wherein the parent interferon is interferon ocl .

54. The hyperglycosylated interferon variant of claim 53, wherein interferon ocl has been modified to include at least one additional glycosylation site, wherein at least one additional glycosylation site is introduced by an amino acid substitution selected from the group consisting of X^X31N, X³33N, X⁴52N, X⁵54T, X⁶72N, X⁹78T, X¹⁰101N, Xⁿ102N, X¹³108N, X¹⁵138T, and X¹⁷145N, wherein X¹ is an Aspartic acid (D), X³ is a Proline (P), X⁴ is a Serine (S), X⁵ is an Isoleucine (I), X⁶ is a Glutamic Acid (E), X⁹ is a Phenylalanine (F), X¹⁰ is a Lysine (K), X¹¹ is an Aspartic acid (D), X¹³ is an Aspartic acid (D), X¹⁵ is a Glycine

(G) , and X¹⁷ is an Alanine (A).

55. The hyperglycosylated interferon variant of any one of claims 29 to 30, wherein the parent interferon is interferon oc4a.

56. The hyperglycosylated interferon variant of claim 55, wherein interferon oc4a has been modified to include at least one additional glycosylation site, wherein at least one additional glycosylation site is introduced by an amino acid substitution selected from the group consisting of X^X31N, X³33N, X⁴52N, X⁵54T, X⁶72N, X⁸76N, X⁹78T, X¹⁰101N, X^U102N, X¹³108N, X¹⁵138T, and X¹⁷145N, wherein X¹ is an Aspartic acid (D), X³ is a Proline (P), X⁴ is a Glycine (G), X⁵ is an Isoleucine (I), X⁶ is a Glutamic Acid (E), X⁸ is a Histidine (H), X⁹ is a Phenylalanine (F), X¹⁰ is a Glutamic acid (E), X¹¹ is an Aspartic acid

(D), X 13 is a Glutamic acid (E), X 15 is a Glutamic acid (E), and X 17 is a Glutamic acid (E).

57. The hyperglycosylated interferon variant of any one of claims 29 to 30, wherein the parent interferon is interferon oc4b.

58. The hyperglycosylated interferon variant of claim 57, wherein interferon oc4b has been modified to include at least one additional glycosylation site, wherein at least one additional glycosylation site is introduced by an amino acid substitution selected from the group consisting of X^X31N, X³33N, X⁴52N, X⁵54T, X⁶72N, X⁸76N, X⁹78T, X¹⁰101N, X^U102N, X¹³108N, X¹⁵138T, and X¹⁷145N, wherein X¹ is an Aspartic acid (D), X³ is a Proline (P), X⁴ is a Glycine (G), X⁵ is an Isoleucine (I), X⁶ is a Glutamic Acid (E), X⁸ is a Histidine (H), X⁹ is a Phenylalanine (F), X¹⁰ is a Glutamic acid (E), X¹¹ is an Aspartic acid

(D), X 13 is a Glutamic acid (E), X 15 is a Glutamic acid (E), and X 17 is a Valine (V).

59. The hyperglycosylated interferon variant of any one of claims 29 to 30, wherein the parent interferon is interferon oc5.

60. The hyperglycosylated interferon variant of claim 59, wherein interferon oc5 has been modified to include at least one additional glycosylation site, wherein at least one additional glycosylation site is introduced by an amino acid substitution selected from the group consisting of X^X31N, X³33N, X⁴52N, X⁵54T, X⁶72N, X⁹78T, X¹⁰101N, Xⁿ102N, X¹³108N, X¹⁵138T, and X¹⁷145N, wherein X¹ is an Aspartic acid (D), X³ is a Proline (P), X⁴ is a Glycine (G), X⁵ is an Isoleucine (I), X⁶ is a Glutamic Acid (E), X⁹ is a Phenylalanine (F), X¹⁰ is a Lysine (K), X¹¹ is an Aspartic acid (D), X¹³ is an Aspartic acid (D), X¹⁵ is a Glutamic acid (E), and X¹⁷ is a Valine (V).

61. The hyperglycosylated interferon variant of any one of claims 29 to 30, wherein the parent interferon is interferon oc7.

62. The hyperglycosylated interferon variant of claim 61, wherein interferon oc7 has been modified to include at least one additional glycosylation site, wherein at least one additional glycosylation site is introduced by an amino acid substitution selected from the group consisting of X^X31N, X³33N, X⁴52N, X⁵54T, X⁶72N, X⁸76N, X⁹78T, X¹⁰101N, X^U102N, X¹³108N, X¹⁵138T, and X¹⁷145N, wherein X¹ is an Aspartic acid (D), X³ is a Proline (P), X⁴ is a Glycine (G), X⁵ is an Isoleucine (I), X⁶ is a Glutamic Acid (E), X⁸ is a Histidine (H), X⁹ is a Phenylalanine (F), X¹⁰ is a Glutamic acid (E), X¹¹ is an Aspartic acid

63. The hyperglycosylated interferon variant of any one of claims 29 to 30, wherein the parent interferon is interferon oc8.

64. The hyperglycosylated interferon variant of claim 63, wherein interferon oc8 has been modified to include at least one additional glycosylation site, wherein at least one additional glycosylation site is introduced by an amino acid substitution selected from the group consisting of X^X31N, X³33N, X⁴52N, X⁵54T, X⁶72N, X⁸76N, X⁹78T, X¹⁰101N, X^U102N, X¹³108N, X¹⁵138T, and X¹⁷145N, wherein X¹ is an Aspartic acid (D), X³ is a Proline (P), X⁴ is a Arginine (R), X⁵ is an Isoleucine (I), X⁶ is a Glutamic Acid (E), X⁸ is a Lysine (K), X⁹ is a Phenylalanine (F), X¹⁰ is a Lysine (K), X¹¹ is an Aspartic acid (D), X¹³ is an Aspartic acid (D), X 15 is an Isoleucine (I), and X 17 is a Glutamic acid (E).

65. The hyperglycosylated interferon variant of any one of claims 29 to 30, wherein the parent interferon is interferon oclO.

66. The hyperglycosylated interferon variant of claim 65, wherein interferon oclO has been modified to include at least one additional glycosylation site, wherein at least one additional glycosylation site is introduced by an amino acid substitution selected from the group consisting of X^X31N, X³33N, X⁴52N, X⁵54T, X⁶72N, X⁹78T, X¹⁰101N, Xⁿ102N, X¹³108N, X¹⁵138T, and X¹⁷145N, wherein X¹ is an Aspartic acid (D), X³ is a Proline (P), X⁴ is a Glycine (G), X⁵ is an Isoleucine (I), X⁶ is a Glutamic Acid (E), X⁹ is a Phenylalanine (F), X¹⁰ is a Glutamic Acid (E), X¹¹ is an Aspartic acid (D), X¹³ is a Glutamic Acid (E), X¹⁵ is a

Glutamic Acid (E), and X 17 is a Glutamic acid (E).

67. The hyperglycosylated interferon variant of any one of claims 29 to 30, wherein the parent interferon is interferon ocl3.

68. The hyperglycosylated interferon variant of claim 67, wherein interferon ocl3 has been modified to include at least one additional glycosylation site, wherein at least one additional glycosylation site is introduced by an amino acid substitution selected from the group consisting of X^X31N, X³33N, X⁴52N, X⁵54T, X⁶72N, X⁹78T, X¹⁰101N, Xⁿ102N, X¹³108N, X¹⁵138T, and X¹⁷145N, wherein X¹ is an Aspartic acid (D), X³ is a Proline (P), X⁴ is a Serine (S), X⁵ is an Isoleucine (I), X⁶ is a Glutamic Acid (E), X⁹ is a Phenylalanine (F), X¹⁰ is a Lysine (K), X¹¹ is an Aspartic acid (D), X¹³ is an Aspartic Acid (D), X¹⁵ is a

Glutamic Acid (E), and X 17 is an Alanine (A).

69. The hyperglycosylated interferon variant of any one of claims 29 to 30, wherein the parent interferon is interferon ocl4.

70. The hyperglycosylated interferon variant of claim 69, wherein interferon ocl4 has been modified to include at least one additional glycosylation site, wherein at least one additional glycosylation site is introduced by an amino acid substitution selected from the group consisting of X³33N, X⁴52N, X⁵54T, X⁶72N, X⁹78T, X¹⁰101N, X¹³108N, X¹⁵138T, and X¹⁷145N, wherein X³ is a Serine (S), X is a Arginine (R), X is an Isoleucine (I), X is a Glutamic Acid (E), X⁹ is a Phenylalanine (F), X¹⁰ is a Lysine (K), X¹¹ is an Aspartic acid (D),

X 15 is a Glutamic Acid (E), and X 17 is an Alanine (A).

71. The hyperglycosylated interferon variant of any one of claims 29 to 30, wherein the parent interferon is interferon ocl6.

72. The hyperglycosylated interferon variant of claim 71, wherein interferon ocl6 has been modified to include at least one additional glycosylation site, wherein at least one additional glycosylation site is introduced by an amino acid substitution selected from the group consisting of X^X31N, X³33N, X⁴52N, X⁵54T, X⁶72N, X⁹78T, X¹⁰101N, Xⁿ102N, X¹³108N, X¹⁵138T, X¹⁷145N, wherein X¹ is an Aspartic acid (D), X³ is a Proline (P), X⁴ is a Glycine (G), X⁵ is an Isoleucine (I), X⁶ is a Valine (V), X⁹ is a Phenylalanine (F), X¹⁰ is a Lysine (K), X¹¹ is an Aspartic acid (D), X¹³ is an Aspartic Acid (D), X¹⁵ is a Glutamic Acid (E), and X¹⁷ is a Glutamic Acid (E).

73. The hyperglycosylated interferon variant of any one of claims 29 to 30, wherein the parent interferon is interferon ocl7.

74. The hyperglycosylated interferon variant of claim 73, wherein interferon ocl7 has been modified to include at least one additional glycosylation site, wherein at least one additional glycosylation site is introduced by an amino acid substitution selected from the group consisting of X^X31N, X³33N, X⁴52N, X⁵54T, X⁶72N, X⁹78T, X¹⁰101N, Xⁿ102N, X¹³108N, X¹⁵138T, and X¹⁷145N, wherein X¹ is an Aspartic acid (D), X³ is a Proline (P), X⁴ is a Glycine (G), X⁵ is an Isoleucine (I), X⁶ is a Glutamic Acid (E), X⁹ is a Phenylalanine (F), X¹⁰ is a Glutamic Acid (E), X¹¹ is an Aspartic acid (D), X¹³ is a Glutamic Acid (E), X¹⁵ is a

Glutamic Acid (E), and X 17 is a Glutamic Acid (E).

75. The hyperglycosylated interferon variant of any one of claims 29 to 30, wherein the parent interferon is interferon oc21.

76. The hyperglycosylated interferon variant of claim 75, wherein interferon oc21 has been modified to include at least one additional glycosylation site, wherein at least one additional glycosylation site is introduced by an amino acid substitution selected from the group consisting of X^X31N, X³33N, X⁴52N, X⁵54T, X⁶72N, X⁹78T, X¹⁰101N, Xⁿ102N, X¹³108N, X¹⁵138T, and X¹⁷145N, wherein X¹ is an Aspartic acid (D), X³ is a Proline (P), X⁴ is a Glycine (G), X⁵ is an Isoleucine (I), X⁶ is a Glutamic Acid (E), X⁹ is a Phenylalanine (F), X¹⁰ is a Lysine (K), X¹¹ is an Aspartic acid (D), X¹³ is a Glutamic Acid (E), X¹⁵ is a Glutamic Acid (E), and X¹⁷ is a Valine (V).

77. The hyperglycosylated interferon variant of any one of claims 29 to 30, wherein the parent interferon is interferon ocH.

78. The hyperglycosylated interferon variant of claim 77, wherein interferon ocH has been modified to include at least one additional glycosylation site, wherein at least one additional glycosylation site is introduced by an amino acid substitution selected from the group consisting of X³33N, X⁴52N, X⁵54T, X⁶72N, X⁹78T, X¹⁰101N, X¹³108N, X¹⁵138T, and X^ HSN, wherein X^J is a Serine (S), X is a Arginine (R), X is an Isoleucine (I), X is a Glutamic Acid (E), X⁹ is a Phenylalanine (F), X¹⁰ is a Lysine (K), X¹³ is an Aspartic Acid

(D), X 15 is a Glutamic Acid (E), and X 17 is a Glutamic Acid (E).

79. The hyperglycosylated interferon variant of any one of claims 29 to 30, wherein the parent interferon is interferon ocl.

80. The hyperglycosylated interferon variant of claim 79, wherein interferon ocl has been modified to include at least one additional glycosylation site, wherein at least one additional glycosylation site is introduced by an amino acid substitution selected from the group consisting of X^X31N, X³33N, X⁴52N, X⁵54T, X⁶72N, X⁹78T, X¹⁰101N, Xⁿ102N, X¹³108N, X¹⁵138T, and X¹⁷145N, wherein X¹ is an Aspartic acid (D), X³ is a Proline (P), X⁴ is a Glycine (G), X⁵ is an Isoleucine (I), X⁶ is a Glutamic Acid (E), X⁹ is a Phenylalanine (F), X¹⁰ is a Glutamic Acid (E), X¹¹ is an Aspartic acid (D), X¹³ is a Glutamic Acid (E), X¹⁵ is a

Glutamic Acid (E), and X 17 is a Glutamic Acid (E).

81. The hyperglycosylated interferon variant of any one of claims 29 to 30, wherein the parent interferon is interferon ocJl.

82. The hyperglycosylated interferon variant of claim 81, wherein interferon odl has been modified to include at least one additional glycosylation site, wherein at least one additional glycosylation site is introduced by an amino acid substitution selected from the group consisting of X^X31N, X³33N, X⁴52N, X⁵54T, X⁶72N, X⁸76N, X⁹78T, X¹⁰101N, X^U102N, X¹³108N, X¹⁵138T, and X¹⁷145N, wherein X¹ is an Aspartic acid (D), X³ is a Proline (P), X⁴ is a Glycine (G), X⁵ is an Isoleucine (I), X⁶ is a Glutamic Acid (E), X⁸ is a Histidine (H), X⁹ is a Phenylalanine (F), X¹⁰ is a Glutamic Acid (E), X¹¹ is an Aspartic acid

83. The hyperglycosylated interferon variant of any one of claims 29 to 30, wherein the interferon a (IFN a) is interferon a6.

84. The hyperglycosylated interferon variant of claim 83, wherein the peptide extension is located between amino acids D29 and C30 of the interferon a, wherein the positions of the amino acid substitutions are with reference to sequence alignment numbering set forth in Figure 1.

85. The hyperglycosylated interferon variant of any one of claims 83 to 84, wherein interferon oc6 has been modified to include at least one additional glycosylation site, wherein at least one additional glycosylation site is introduced by an amino acid substitution selected from the group consisting of X^X31N, X³33N, X⁴52N, X⁵54T, X⁶72N, X⁹78T, X¹⁰101N, X^U102N, X¹³108N, X¹⁵138T, and X¹⁷145N, wherein X¹ is an Aspartic acid (D), X³ is a Proline (P), X⁴ is an Arginine (R), X⁵ is an Isoleucine (I), X⁶ is a Glutamic Acid (E), X⁹ is a Phenylalanine (F), X¹⁰ is a Glutamic Acid (E), X¹¹ is an Aspartic acid (D), X¹³ is an

Aspartic Acid (D), X 15 is a Glycine (G), and X 17 is a Glutamic Acid (E).

86. The hyperglycosylated interferon variant of any one of claims 25 to 26, wherein the parent interferon is interferon β.

87. The hyperglycosylated interferon variant of claim 86, wherein the peptide extension is inserted between amino acids S27 and M28 of interferon β, wherein the positions of the amino acid substitutions are with reference to sequence alignment numbering set forth in Figure 1.

88. The hyperglycosylated interferon variant of any one of claims 86 to 87, wherein interferon β has been modified to include at least one additional glycosylation site, wherein at least one additional glycosylation site is introduced by an amino acid substitution selected from the group consisting of X³33T, X³33N, X⁵54T, X⁶72N, X⁸76N, X⁹78T, X¹⁰101N, X^U102N, and X¹⁵138T, wherein X³ is a Leucine (L), X⁴ is an Arginine (R), X⁶ is an Isoleucine (I), X⁸ is a Glutamine (Q), X⁹ is a Phenylalanine (F), X¹⁰ is an Aspartic Acid (D), X¹¹ is a Serine (S), and X¹⁵ is a Glutamic acid (E).

89. The hyperglycosylated interferon variant of claim 88, wherein at least one additional glycosylation site is introduced to interferon β by the amino acid substitution

X 333T, wherein X 3 is a Leucine (L).

90. The hyperglycosylated interferon variant of claim 89, further comprising the amino acid substitution X 232S, wherein X 2 is a Leucine (L).

91. The hyperglycosylated interferon variant of claim 88, wherein at least one additional glycosylation site is introduced to interferon β by the amino acid substitution substitution X 333N, wherein X 3 is a Leucine (L).

92. The hyperglycosylated interferon variant of claim 91, further comprising the amino acid substitution F35T.

93. The hyperglycosylated interferon variant of claim 88, wherein at least one additional glycosylation site is introduced to interferon β by the amino acid substitution X⁴54T, wherein X⁴ is an Arginine (R).

94. The hyperglycosylated interferon variant of claim 88, wherein at least one additional glycosylation site is introduced to interferon β by the amino acid substitution X⁶72N, wherein X⁶ is an Isoleucine (I).

95. The hyperglycosylated interferon variant of claim 94, further comprising the amino acid substitution X 774T, wherein X 7 is a Glutamine (Q).

96. The hyperglycosylated interferon variant of claim 88, wherein at least one additional glycosylation site is introduced to interferon β by the amino acid substitution X⁹78T, wherein X⁹ is a Phenylalanine (F).

97. The hyperglycosylated interferon variant of claim 88, wherein at least one additional glycosylation site is introduced to interferon β by the amino acid substitution X^1U101N, wherein X^u is an Aspartic (D).

98. The hyperglycosylated interferon variant of claim 88, wherein at least one additional glycosylation site is introduced to interferon β by the amino acid substitution X^U102N, wherein X¹¹ is a Serine (S).

99. The hyperglycosylated interferon variant of claim 98, further comprising the amino acid substitution X 12104T, wherein X 12 is a Serine (S).

100. The hyperglycosylated interferon variant of claim 88, wherein at least one additional glycosylation site is introduced to interferon β by the amino acid substitution X°138T, wherein X¹¹ is a Glutamic acid (E).

101. The hyperglycosylated interferon variant of claim any one of claims 25 to 26, wherein the parent interferon is interferon κ.

102. The hyperglycosylated interferon variant of claim 101, wherein interferon κ has been modified to include at least one additional glycosylation site, wherein at least one additional glycosylation site is introduced by an amino acid substitution selected from the group consisting of X³33N, X⁴52N, X⁵54T, X⁶72N, X⁸76N, X⁹78T, X¹⁰101N, Xⁿ102N, X¹³108N, X¹⁵138T, and X¹⁷145N, wherein X³ is a Leucine (L), X⁴ is a Serine (S), X⁵ is a Serine (S), X⁶ is a Phenylalanine (F), X⁸ is a Threonine (T), X⁹ is a Proline (P), X¹⁰ is a Histidine (H), X¹¹ is a Threonine (T), X¹³ is a Lysine (K), X¹⁵ is a Glutamic acid (E), and X¹⁷ is a Glutamic Acid (E).

103. The hyperglycosylated interferon variant of claim any one of claims 25 to 26, wherein the parent interferon is interferon ω.

104. The hyperglycosylated interferon variant of claim 103, wherein interferon ω has been modified to include at least one additional glycosylation site, wherein at least one additional glycosylation site is introduced by an amino acid substitution selected from the group consisting of X^X31N, X³33N, X⁴52N, X⁵54T, X⁶72N, X⁸76N, X⁹78T, X¹⁰101N, X^U102N, X¹⁵138T, and X¹⁷145N, wherein X¹ is an Aspartic acid (D), X³ is a Proline (P), X⁴ is an Arginine (R), X⁵ is an Isoleucine (I), X⁶ is a Methionine (M), X⁸ is a Serine (S), X⁹ is a Leucine (L), X¹⁰ is a Glutamic acid (E), X¹¹ is an Arginine (R), X¹⁵ is a Glycine (G), and X¹⁷ is a Serine (S).

105. The hyperglycosylated interferon variant of claim any one of claims 25 to 26, wherein the parent interferon is interferon x.

106. The hyperglycosylated interferon variant of claim 105, wherein interferon x has been modified to include at least one additional glycosylation site, wherein at least one additional glycosylation site is introduced by an amino acid substitution selected from the group consisting of X^X31N, X³33N, X⁴52N, X⁵54T, X⁶72N, X⁸76N, X⁹78T, Xⁿ102N, X¹³108N, X¹⁵138T, and X¹⁷145N, wherein X¹ is a Lysine (K), X³ is an Isoleucine (I), X⁴ is an Glutamine (Q), X⁵ is a Leucine (L), X⁶ is a Serine (S), X⁸ is a Glutamine (Q), X⁹ is a Tyrosine (Y), X¹¹ is an Isoleucine (I), X¹³ is a Glutamic acid (E), X¹⁵ is a Glutamic acid (E), and X¹⁷ is a Glycine (G).

107. The hyperglycosylated interferon variant of any one of claims 1 to 23, wherein the parent interferon is a Type II interferon.

108. The hyperglycosylated interferon variant of claim 107, wherein the Type II interferon is interferon γ (IFN γ).

109. The hyperglycosylated interferon variant of claim 108, wherein the peptide extension is inserted between amino acids G20 and C21 of interferon γ.

110. The hyperglycosylated interferon variant of any one of claims 108 to 109, wherein interferon γ has been modified to include at least one additional glycosyation site, wherein at least one additional glycosylation site is introduced by an amino acid substitution selected from the group consisting of D25N and V28N.

111. The hyperglycosylated interferon variant of claim 110, wherein at least one additional glycosyation site is introduced to interferon γ by the amino acid substitution D25N.

112. The hyperglycosylated interferon variant of claim 111, further comprising the amino acid substitutions P26S+Y27T.

113. The hyperglycosylated interferon variant of claim 110, wherein at least one additional glycosyation site is introduced to interferon γ by the amino acid substitution V28N.

114. The hyperglycosylated interferon variant of claim 113, further comprising the amino acid substitutions K29S+E30T.

115. The hyperglycosylated interferon variant of any one of claims 1 to 23, wherein the parent interferon is a Type III interferon.

116. The hyperglycosylated interferon variant of claim 115, wherein the Type III interferon is selected from the group consisting of interferon λΐ, interferon λ2, and interferon λ3.

117. The hyperglycosylated interferon variant of claim 116, wherein the Type III interferon is interferon λΐ.

118. The hyperglycosylated interferon variant of claim 117, wherein the peptide extension is inserted between amino acids A19 and G20 of interferon λΐ.

119. The hyperglycosylated interferon variant of any one of claims 117 to 118, wherein interferon λΐ has been modified to include at least one additional glycosyation site, wherein at least one additional glycosylation site is introduced by an amino acid substitution selected from the group consisting of P21N and P27N.

120. The hyperglycosylated interferon variant of claim 119, wherein at least one additional glycosylation site is introduced to interferon λΐ by the amino acid substitution P21N.

121. The hyperglycosylated interferon variant of claim 120, further comprising the amino acid substitutions selected V22S+P23T.

122. The hyperglycosylated interferon variant of claim 119, wherein at least one additional glycosylation site is introduced to interferon λΐ by the amino acid substitution P27N.

123. The hyperglycosylated interferon variant of claim 122, further comprising the amino acid substitution T28S.

124. The hyperglycosylated interferon variant of claim 116, wherein the Type III interferon is interferon λ2.

125. The hyperglycosylated interferon variant of claim 124, wherein the peptide extension is inserted between amino acids A25 and V26 of interferon λ2.

126. The hyperglycosylated interferon variant of any one of claims 124 to 125, wherein interferon λ2 has been modified to include at least one additional glycosyation site, wherein at least one additional glycosylation site is introduced by an amino acid substitution selected from the group consisting of P27N and G33N.

127. The hyperglycosylated interferon variant of claim 126, wherein at least one additional glycosylation site is introduced to interferon λ2 by the amino acid substitution P27N.

128. The hyperglycosylated interferon variant of claim 127, further comprising the amino acid substitutions V28S+A29T.

129. The hyperglycosylated interferon variant of claim 126, wherein at least one additional glycosylation site is introduced to interferon λ2 by the amino acid substitution G33N.

130. The hyperglycosylated interferon variant of claim 129, further comprising the amino acid substitutions A34S+L35T.

131. The hyperglycosylated interferon variant of claim 116, wherein the Type III interferon is interferon λ3.

132. The hyperglycosylated interferon variant of claim 131, wherein the peptide extension is inserted between amino acids A21 and V22 of interferon λ3.

133. The hyperglycosylated interferon variant of any one of claims 131 to 132, wherein interferon λ3 has been modified to include at least one additional glycosyation site, wherein at least one additional glycosylation site is introduced by an amino acid substitution selected from the group consisting of P23N and G29N.

134. The hyperglycosylated interferon variant of claim 133, wherein at least one additional glycosylation site is introduced to interferon λ3 by the amino acid substitution P23N.

135. The hyperglycosylated interferon variant of claim 134, further comprising the amino acid substitutions V24S+A25T.

136. The hyperglycosylated interferon variant of claim 133, wherein at least one additional glycosylation site is introduced to interferon λ3 by the amino acid substitution G29N.

137. The hyperglycosylated interferon variant of claim 136, further comprising the amino acid substitutions A30S+L31T.

138. A pharmaceutical composition comprising the hyperglycosylated interferon variant of any one of claims 1-137; and a pharmaceutically acceptable excipient.

139. The pharmaceutical composition of claim 138, wherein the pharmaceutically acceptable excipient is suitable for oral delivery.

140. The pharmaceutical composition of claim 138, wherein the pharmaceutically acceptable excipient is suitable for parenteral delivery.